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She anchored the victorious United States team in the 4x100m relay with a winning time of 44.5 seconds - slightly outside the world record of 44.4 set by the US team in the semi-finals.
Rudolph, 20, has already won the 100m gold in a wind-assisted time of 11.0 seconds and took the 200m in a time of 24 seconds - after setting an Olympic record of 23.2 in her opening heat.
She is the first American woman athlete to win three track and field gold medals at one Olympic Games.
Rudolph, a striking figure at 6ft (1.8m) tall, first ran in the Melbourne Olympics. Although she was eliminated in the 200m heats, she did win a bronze medal in the 4x100m relay.
The day before the 100m in Rome, she stepped in a hole at the practice track and twisted her ankle. Despite having it strapped up, she still won her semi-final in a world-record equalling 11.3 seconds.
Her 100m final time of 11.0 seconds would have been a world record, but the following wind of 6.15 miles an hour (9.9kmh) meant it was disallowed.
Today's third gold in the relay event was thrilling to watch. The US team were lying in second place with a two yard (1.8m) deficit after a bad baton pass, but Rudolph closed the gap and went on to win by three clear yards (2.7m).
Rudolph was born in Clarksville, Tennessee. She was the 20th of 22 children and weighed just four-and-a-half pounds (2kg) at birth.
At the age of four, she contracted double pneumonia, scarlet fever and polio and almost died. The illnesses left her left leg paralysed and for several years she had to wear a brace.
Doctors said daily massage might help repair her wasted leg muscle and with plenty of family help by the age of nine Rudolph was able to get by with an orthopaedic shoe.
Two years later Rudolph had abandoned her shoe. She began playing basketball and became increasingly competitive.
She was spotted by athletics coach Ed Temple at Tennessee State University who invited her to his summer training camps and subsequently picked her to run in the Melbourne Olympics.
She was awarded an athletic scholarship at Tennessee State and became part of Temple's Tigerbelles track team, returning to the Olympics in Rome as a medal favourite.
Wilma Rudolph set several world sprint records after Rome - but retired aged just 22 in 1962.
She said at the time she wanted to be remembered at her best and not as an athlete in decline.
She became an American hero and also did much for the emancipation of black women in the US.
With athletics still a strictly amateur sport, however, Rudolph was unable to profit from her fame and went on to make a living as a schoolteacher and sports coach.
She was elected to the National Track and Field Hall of Fame in 1974. In 1993 she was also one of the first five people to receive a new national sports award and dinner at the White House in 1993.
She also became US ambassador of goodwill to French West Africa and she formed her own non-profit making organisation, the Wilma Rudolph foundation, to help underprivileged children.
She died in 1994 from brain cancer, leaving two sons and two daughters.
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