Egg incubation temperature controls sexual differentiation of the gonads in the saltwater crocodile, Crocodylus porosus. Embryos incubated at 30°C develop ovaries and hatch as females while most embryos incubated at 32–32.5°C develop testes and hatch as males. Several lines of evidence suggest that estrogen is involved in gonadal sex differentiation in crocodilians and other reptiles with temperature‐dependent sex determination (TSD). In vivo autoradiography was used here to identify sites of 3H‐estradiol uptake in the urogenital tissues of C. porosus embryos incubated at 30°C (female‐producing) and at 32.5°C (predominantly male‐producing). During the period of gonadal differentiation (developmental stage 23), 3H‐estradiol was specifically concentrated in the gonads of presumptive females at 30°C and presumptive males at 32.5°C. Binding was localised primarily in the medulla of the gonad in both sexes, with little or no uptake in the cortex. No uptake was detected in the adjoining adrenal glands, at either temperature, although some uptake was observed in the mesonephros at the female temperature (30°C). No specific binding of estradiol by the gonads was found prior to (stage 19) or after (stage 28) the period of gonadal differentiation. These results are consistent with the hypothesis that estrogen binding sites are present in the gonads of both sexes during a specific period of embryogenesis, which coincides with the time of gonadal sex differentiation. Furthermore, these observations suggest that the role of estrogen in TSD is not mediated through differential uptake of the steroid by presumptive ovaries and testes. It is therefore hypothesised that estrogen synthesis is temperature‐sensitive, rather than estrogen binding by the gonad. © 1994 Wiley‐Liss, Inc.