Understanding the proximate mechanism for seasonal modulation of the adrenocortical stress response is an important step in determining the evolutionary significance of such modulation. In vitro studies of adrenocortical cell function in Eastern Fence Lizards (Sceloporus undulatus) have partially explained the mechanism for seasonal changes in male stress responsiveness, but studies of free-living females have not been conducted. Here we test two predictions generated from the in vitro work in free-living female fence lizards: the plasma corticosterone response to adrenocorticotropic hormone injection will be weaker in the postbreeding than the breeding season, and baseline corticosterone levels will be lower in postbreeding than breeding females. Twice during the breeding season and once during the postbreeding season, corticosterone concentrations were measured at capture then again after adrenocorticotropic hormone injection and one hour of confinement. Females robustly responded to adrenocorticotropic hormone, but there was no detectable seasonal difference in the strength of the corticosterone response. Similarly, corticosterone responses did not vary seasonally in females subjected only to confinement without injection. However, as predicted, baseline plasma corticosterone levels were lower in the postbreeding than the breeding season. Plasma triglyceride concentrations varied with season as well as being negatively associated with baseline corticosterone levels. The whole-organism corticosterone response may differ from that of isolated adrenocortical cells which are not subject to either paracrine or upstream hypothalamic and pituitary signals. The mechanism of adrenocortical modulation and its evolutionary significance is most likely to be deciphered by conducting studies at multiple levels of organization.