Reduced reproductive success, altered reproductive tract development, and differences in circulating hormones have been documented in American alligators (Alligator mississippiensis) from Lake Apopka, FL, compared to less contaminated sites, such as the Lake Woodruff National Wildlife Refuge, FL. Comparative studies among alligators of varying size and age suggest that in ovo contaminant-induced alterations of endocrine function are further modified during postembryonic development and/or through environmental exposure. In the present study, we examined developmental and endocrine-related indices in neonatal (age, <1 mo) alligators from Lake Apopka in comparison to those of a reference population (Lake Woodruff), thereby limiting contaminant exposure to that derived via maternal contribution. We compared several reproductive and developmental parameters, including hatching success, primary sex determination, and somatic indices. Furthermore, we examined circulating testosterone concentrations and aromatase activity in an effort to establish relative gonadal endocrine function shortly after hatching. Finally, we compared phallus size among males and oviduct epithelial cell height (ECH) among females (androgen- and estrogen-dependent tissues, respectively). Significant differences between populations were noted for body size and spleen somatic index. Neonatal alligators from Lake Apopka exhibited higher plasma testosterone, but no differences were detected in gonadal aromatase activity compared to Lake Woodruff. Phallus tip length and cuff diameter were smaller in males from Lake Apopka, whereas no differences were noted in oviduct ECH. Our data establish basic indices of development and endocrine function in neonatal alligators before environmental exposure to contaminants. These results should begin to help separate developmental abnormalities resulting from in ovo exposure, presumably of maternal origin, from physiological alterations induced through environmental exposure to contaminants.