The rat snake, the main attraction at the World Aquarium in St Louis, US, died of natural causes during the weekend.
Most two-headed snakes survive for only a week or two.
"We", the two-headed snake, died after an impressive eight years in a US aquarium
Caretaker Leonard Sonnenschein said: "It's terrible news. People come in every day and say: 'I'm here to see the two-headed snake.'"Sonnenschein said more than a million people have seen We over the years.
Children were especially fascinated, wondering how two heads could coexist on the same body as We sometimes strained to slither in two directions at once.
"These kinds of questions helped spur the science spirit in children," Sonnenschein said.
The snake had both male and female genitalia, Sonnenschein said.
The aquarium tried unsuccessfully to breed We with another two-headed snake last year, and had planned to try again this summer, aquarium spokesman David Isserman said.
A taxidermist is preserving his body, which should be back on display within a week, Sonnenschein said.
The World Aquarium acquired We in 1999. Last year, the aquarium tried to auction off the rare reptile to raise money for research and conservation programs, asking £75,000, but none of the bids was satisfactory.
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