The factors controlling sexual differentiation in crocodilians are unknown, but heteromorphic sex chromosomes are absent from all species1. Nichols and Chabreck2 speculated that the sex of Alligator mississippiensis was not rigidly determined at the time of hatching but could be influenced by the post-hatching environment. They presented little evidence to support their hypothesis3 (no histological sections of hatchling gonads, no indication of the sex ratio at hatching), and their study failed to take account of habitat preferences of adult male and female alligators4. Here we demonstrate by laboratory and field experiments, that in A. mississippiensis: (1) Sex is fully determined at the time of hatching and naturally irreversible thereafter, and depends on the temperature of egg incubation, temperatures ⩽30 °C producing all females, ⩾34 °C yielding all males. (2) The temperature-sensitive period is between 7 and 21 days of incubation. (3) Natural nests constructed on levees are hotter (34 °C) than those constructed on wet marsh (30 °C), thus the former hatch males and the latter females. (4) The natural sex ratio at hatching is five females to 1 male. (5) Females hatched from eggs incubated at 30 °C weigh significantly more than males hatched from eggs incubated at 34 °C. This weight difference constitutes a possible selective evolutionary advantage of temperature-dependent sex determination (TSD) in alligators in that females become large and sexually mature as early as possible. The occurrence of TSD in alligators has wide-ranging implications for embryological, teratological, molecular, evolutionary, conservation and farming studies as well as for theories relating to the extinction of other Archosaurs.