Tyson describes the discovery of cosmic rays by Victor Hess through high-altitude balloon trips. Swiss Astronomer Fritz Zwicky, in studying supernovae, postulated that these cosmic rays originated from these events instead of electromagnetic radiation. Also tells how Vera Rubin observed that the rotation of stars at the edges of observable galaxies did not follow expected rotational behavior leading to consider the existence of dark matter. This further led to the discovery of dark energy to account for the increasing rate of expansion of the universe. Tyson then describes the interstellar travel of the two Voyager probes. Tyson tells the Carl Sagan's role in the Voyager program, including creating the Voyager Golden Record to encapsulate humanity and Earth's position in the universe. Tyson concludes the series by emphasizing Sagan's message on the human condition in the vastness of the cosmos, and to encourage viewers to continue to explore and discover what else the universe has to offer.

Sagan discusses the story of the Heike crab and artificial selection of crabs resembling samurai warriors, as an opening into a larger discussion of evolution through natural selection (and the pitfalls of intelligent design). Among the topics are the development of life on the Cosmic Calendar and the Cambrian explosion; the function of DNA in growth; genetic replication, repairs, and mutation; the common biochemistry of terrestrial organisms; the creation of the molecules of life in the Miller-Urey experiment; and speculation on alien life (such as life in Jupiter's clouds). In the Cosmos Update ten years later, Sagan remarks on RNA also controlling chemical reactions and reproducing itself and the different roles of comets (potentially carrying organic molecules or causing the Cretaceous–Paleogene extinction event).

Beginning with the separation of the fuzzy thinking and pious fraud of astrology from the careful observations of astronomy, Sagan follows the development of astronomical observation. Beginning with constellations and ceremonial calendars (such as those of the Anasazi), the story moves to the debate between Earth and Sun-centered models: Ptolemy and the geocentric worldview, Copernicus' theory, the data-gathering of Tycho Brahe, and the achievements of Johannes Kepler (Kepler's laws of planetary motion and the first science-fiction novel).

Ideas about time and space are explored in the changes that constellations undergo over time, the redshift and blue shift measured in interstellar objects, time dilation in Albert Einstein's theory of relativity, the designs of both Leonardo da Vinci and spacecraft that could travel near light speed, time travel and its hypothetical effects on human history, the origins of the Solar System, the history of life, and the immensity of space. In Cosmos Update, the idea of faster-than-light travel by wormholes (researched by Kip Thorne and shown in Sagan’s novel Contact) is discussed.

Beginning with the origins of the universe in the Big Bang, Sagan describes the formation of different types of galaxies and anomalies such as galactic collisions and quasars. The episodes moves further into ideas about the structure of the Universe, such as different dimensions (in the imaginary Flatland and four-dimensional hypercubes), an infinite vs. a finite universe, and the idea of an oscillating Universe (similar to that in Hindu cosmology). The search into other ideas such as dark matter and the multiverse is shown, using tools such as the Very Large Array in New Mexico. Cosmos Update shows new information about the odd, irregular surfaces of galaxies and the Milky Way perhaps being a barred spiral galaxy

Carl Sagan teaches students in a classroom in his childhood home in Brooklyn, New York, which leads into a history of the different mythologies about stars and the gradual revelation of their true nature. In ancient Greece, some philosophers (Aristarchus of Samos, Thales of Miletus, Anaximander, Theodorus of Samos, Empedocles, Democritus) freely pursue scientific knowledge, while others (Plato, Aristotle, and the Pythagoreans) advocate slavery and epistemic secrecy.