The egg yolk serves as a significant source of maternally derived steroids that are available to the embryo during early development. Altered deposition of yolk steroids can change the developmental trajectory of the embryo and have long lasting or permanent consequences. Alligators from contaminated environments have shown significant reproductive and developmental dysfunction, and it is unclear if altered deposition of yolk steroids could be a contributing factor. Alligator eggs were collected from Lake Woodruff (a reference lake), Lake Apopka (a site of known agricultural contamination), and the Merritt Island National Wildlife Refuge (MINWR) (home of the Kennedy Space Center and a site of heavy metal contamination). The yolks of eggs at embryonic stages 12 (prior to sex determination) and 24 (post-sex determination) were evaluated for concentrations of progesterone, 17-beta estradiol, and testosterone. Yolk concentrations of progesterone were significantly lower at embryonic stage 12 in eggs from Lake Apopka and MINWR when compared to eggs from Lake Woodruff. Yolk concentrations of 17-beta estradiol were significantly lower at embryonic stage 12 in eggs from MINWR when compared to the other two sites. Reductions in yolk 17-beta estradiol concentrations from embryonic stage 12 to 24 were significantly attenuated in eggs from MINWR versus that of Lakes Woodruff and Apopka. This study suggests that altered deposition of yolk steroids, and possibly differential utilization by the embryo, could be a contributory mechanism in the reproductive and developmental abnormalities seen in alligators from contaminated locales.