Natalia Romanova, a descendant of Russia's royal family, was found as a young child by a Russian soldier known as Ivan Petrovitch, who watched over her as she grew up and today works as her chauffeur. Romanova proved to be a brilliant scholar and athlete as she matured, and in her teens gained fame in the Soviet Union as a ballerina. She married the renowned Soviet test pilot Alexi Shostakov. It was decided at the KGB, the department of the Soviet government, which served as its intelligence agency and secret police, that the Shostakovs would make good special operatives. While Alexi Shostakov was away on a mission, he was informed of the state's new plans for him, and told that from then on he could have no contact with any of his past friends and acquaintances, including even his own wife. Shostakov was then trained to become the Red Guardian, a costumed agent who was intended to act as the Soviet Union's counterpart to Captain America. Meanwhile, a Soviet official told Natalia Shostakova that her husband had died in the explosion of an experimental rocket he was testing. Distraught, Natalia Shostakova said that she wanted to do something to be worthy of the memory of her heroic husband. The KGB had anticipated her reaction, and trained her to become the spy known as the Black Widow. Some theorize that Romanova was trained in the nearly mythical location known as the "Red Room."

At first the Black Widow did not act as a costumed agent. Her first major field assignment was to infiltrate Stark Industries in the United States and to assist her partner Boris Turgenov in the assassination of the defector Professor Anton Vanko. The Black Widow was thwarted repeatedly by the original Iron Man in her various schemes against Stark Industries. The Black Widow used Hawkeye, who was then sought as a criminal and who found himself attracted to her, as an ally in some of her subversive activities. The Widow was given her first costume in her fourth major mission in the United States. However, her growing love for Hawkeye led to a weakening of her resolve to continue her missions for the Soviets. Hawkeye refused to act as a criminal any longer, and successfully sought amnesty and Avengers membership.

The Black Widow fell into the hands of intelligence agents of the People's Republic of China, who brainwashed her into serving them. They directed her to attack the Avengers, but she freed herself from the brainwashing, was reunited with Hawkeye, and defected to the United States. She publicly revealed her true identity and offered her services to S.H.I.E.L.D., which has made use of them on numerous occasions. Her romance with Hawkeye ended, as did her subsequent love affair and partnership with Daredevil, although she remains a friend of both men. She was a member of the Champions of Los Angeles, and serves from time to time as a member of the Avengers. She remains active as a freelance adventurer.