Temperature‐dependent sex determination (TSD) was first described for a crocodilian in Alligator mississippiensis, in which egg incubation at 33°C produces 100% male hatchlings and incubation at 30°C produces 100% females. Ultrastructural signs of testis differentiation in embryos incubated at 33°C are first detected during days 28–34 (developmental stages 21–22), when small numbers of enlarged “presumptive” Sertoli cells (pre‐Sertoli cells) appear in the gonadal medulla of most embryos. These pre‐Sertoli cells proliferate and differentiate into Sertoli cells during stage 23 at 33°C (days 35–41), marking seminiferous cord formation. At 30°C, some pre‐Sertoli‐like cells are also present in the medulla, but they are less numerous than at 33°C and they do not differentiate into Sertoli cells. Ovary differentiation at 30°C is characterized by proliferation of cortical germs cells from day 39 (stage 22), closely followed by proliferation of somatic (pre‐follicular) cells in the cortex and regression of the medullary cords from day 43 (stage 23). We hypothesize that TSD in the alligator might be a function of a temperature‐derived mismatch between Sertoli cell differentiation and germ cell/cortex proliferation. © 1994 Wiley‐Liss, Inc.