In terms of steroid effector systems, one of the areas least explored is the often described species difference in sensitivity to administered glucocorticoids. We have compared a species reported to be corticosensitive (mouse) with a more corticoresistant species (guinea pig) in terms of 1) the affinity with which tritiated dexamethasone ([3H]DM) is bound in spleen cytoplasmic preparations from adrenalectomized animals, 2) the sensitivity to DM of phytohemagglutinin-treated spleen cells in vitro, measured by inhibition of tritiated thymidine incorporation, and 3) the effect of adrenalectomy 1 day before sacrifice on the spleen cell response to DM in vitro. The affinity of mouse spleen glucocorticoid receptors for [3H]DM is represented by a Kd at 4 C of ∼5 nM; in the guinea pig, the same tissue has a 20- fold lower affinity (Kd at 4 C, ∼0.1 μM). The concentrations of DM necessary for half-maximal inhibition of tritiated thymidine incorporation (mouse, 5 nM; guinea pig, 0.08 μM) closely approximate the half-maximal values for glucocorticoid receptor occupancy. Adrenalectomy the day before sacrifice does not significantly change the concentration of DM required for half-maximal inhibition, but is followed by a significant increase in the slope of the dose-response curve measured around the point of 50% inhibition. Conclusions are: 1) there is an ∼20-fold difference in affinity for [3H]DM between glucocorticoid receptors in the two species; 2) this difference in affinity is a sufficient, although not necessarily exclusive, explanation for the demonstrated differences in glucocorticoid sensitivity between the two species; 3) in both species, receptor-effector events appear closely coupled, evidenced by strikingly parallel dose-response curves; and 4) the mechanism whereby prior adrenalectomy increases the slope of the dose-response curve remains to be established.