Wild alligators exposed to persistent organochlorine contaminants, municipal waste compounds, and contemporary-use herbicides exhibit reproductive alterations that are thought to be caused by endocrine disruption. This study tests the hypothesis that these alterations, at least in part, result from exposure of alligator embryos to contemporary-use herbicides. Alligator eggs were collected early in development, exposed to estradiol-17 beta, atrazine, or 2,4-D (at dosages of 0.14, 1.4, and 14 ppm, plus a dosage of 0.014 ppm for estradiol-17 beta only) before the period of gonadal differentiation, and incubated at a temperature that would produce either 100% males or 100% females. Analysis of histology was performed on the gonads and reproductive tracts of hatchlings. In females, epithelial cell height of the Müllerian duct and medullary regression of the ovary were assessed, whereas in males, sex-cord diameter was measured. Eggs incubated at the female-determining temperature produced all female hatchlings, whereas the estradiol-17 beta treatments caused the production of females at the male-determining temperature. Neither atrazine nor 2,4-D had this effect. Both Müllerian duct epithelial cell height and medullary regression were increased in estradiol-treated animals, but no differences were noted between herbicide-treated alligators and controls. A previous study found that male alligators exposed to 14 ppm atrazine had elevated gonadal aromatase activity, but there was no difference in sex-cord diameter in this or any other treatment group. Additionally, we observed that hepatic aromatase activity was not altered by in ovo exposure to any of the treatments. These results indicate that these herbicides alone are not responsible for the gonadal abnormalities previously reported for juvenile alligators from Lake Apopka and emphasize the importance of analyzing both the function (i.e., steroidogenic enzyme activity) and the structure (i.e., histological analysis) of the reproductive system. Structural assessment alone may be insufficient for detecting subtle endocrine alterations.