The series' pilot episode features an accident in which Central City Police forensic scientist Barry Allen's crime lab is struck by lightning. Allen's electrified body is flung into and shatters a cabinet of chemicals, which are both electrified and forced to interact with each other and with his physiology when they come into physical contact with his body. He soon discovers with the help of S.T.A.R. Labs scientist Tina McGee that the accident has changed his body's metabolism and as a result he has gained the ability to move at superhuman speed. To avenge the murder of his brother, motorcycle police officer Jay (named after the Golden Age Flash), Barry demands that Tina modify a red S.T.A.R. Labs prototype deep sea diving suit, designed to withstand tremendous pressures, into his costume, to which she reluctantly complies. Thus, Barry Allen becomes The Flash.

The series initially had a dark and gritty tone, and focused on having The Flash confront decidedly human villains, like corrupt officials and mobsters. Midway through the show's run, however, a few of The Flash's familiar "Rogues Gallery" of colorful super-villains began making appearances.

The most famous Rogues in the series were The Trickster, played by Mark Hamill, and his sidekick Prank, played by Corinne Bohrer. This foreshadowed Hamill's subsequent success at playing The Joker in Batman: The Animated Series. Hamill would later reprise the role of The Trickster on an episode of Justice League Unlimited. Captain Cold, played by Michael Champion, and the Mirror Master, played by David Cassidy as a disgraced expert in holograms, also appeared in their own episodes. Although fans were pleased to see more DC character's inclusions in the series, the interpretations were radically different from the source material, with the exception of Hamill's Trickster. Captain Cold, for instance, was turned into an albino hitman who murdered his victims by literally freezing them to death, while the Mirror Master was little more than a common thug with a nickname.

Ultimately, these appearances were too little, too late to save the show, which struggled with a high per-episode price tag, stiff competition from NBC's strong Thursday night lineup and Fox, and frequent preemptions due to breaking coverage of the Persian Gulf War and NBA basketball games, resulting in low ratings, a fluctuating time slot which resulted a shift to Saturdays by the spring, and eventually, cancellation after a single season.

The series had certain recurring themes/running gags, such as Barry secretly using his superhuman speed to his or others's benefit--both in and out of uniform--moving so fast as to be unseen, and the corresponding surprised reactions of others when things seemed to move or happen with no visible cause.

Another recurring theme was the fact that a side-effect of Barry's powers was a ravenous hunger, due to his body requiring food to replace the tremendous amount of energy he used at super-speed; he would often be shown consuming huge meals, as well as sneaking snacks whenever he could, sometimes at super-speed, such as consuming an entire tray of hors d'œuvres at a reception when no one was looking, or drinking an entire gallon jug of milk in less than five seconds, using enough suction to crush the jug when it emptied. In the recently relaunched The Flash comic book series, the main character was described as having the same metabolic limitation.