Glucocorticoids induce a series of profound biochemical changes in thymocytes that initiate apoptosis; however, the pathways beyond receptor transactivation that lead to this form of cell death are not fully understood. In this study, we report a novel site of action for glucocorticoids at the site of the plasma membrane. Specifically, we find that glucocorticoids induce the loss of plasma membrane potential both in vivo and in vitro. The glucocorticoid-induced loss of plasma membrane potential in cultured primary isolated rat thymocytes was both dose and time dependent. Other steroid hormones, including progesterone, estrogen, and testosterone, fail to alter the depolarization state of the thymocyte plasma membrane. Interestingly, other nonsteroid stimuli that also activate apoptosis in thymocytes also lead to cellular depolarization. In contrast, HeLa cells, which contain functional glucocorticoid receptors but do not die in response to hormone, do not alter their plasma membrane potential in response to glucocorticoids, indicating a strong association between depolarization and apoptosis. Furthermore, the ability of glucocorticoids to depolarize the plasma membrane of thymocytes required the interaction of glucocorticoids with their cognate receptor, because RU486 failed to depolarize thymocytes and antagonized the effect of glucocorticoids. Finally, experiments using inhibitors of transcription and translation indicated that the loss of plasma membrane potential in thymocytes following glucocorticoid treatment required de novo gene expression. The results of these studies establish that the loss of plasma membrane potential is an early important feature of glucocorticoid-induced apoptosis of thymocytes.