Sex hormones have been implicated in the pathogenesis of many autoimmune disorders, presumably through regulatory influences on the immune system. However, the mechanisms of sex steroid action on humoral and cellular immune responses are not precisely understood. In this study, the in vitro effects of physiologic concentrations of 17 beta-estradiol and testosterone on the Ag non-specific differentiation of human PBMC were examined using optimal and sub-optimal doses, respectively, of PWM. In cultures of PBMC from 14 normal donors (7 men and 7 women, aged 25 to 45 yr), 17 beta-estradiol (0.5 to 30 ng/ml) enhanced PWM-induced generation of PFC by 46% (p less than 0.01), whereas testosterone (10 to 300 ng/ml) inhibited PFC generation by a mean of 36% (p less than 0.001). The enhancing and suppressing effects of the sex steroids on PBMC occurred early inasmuch as estradiol and testosterone had to be added to the cultures at their initiation (6 and 24 h, respectively) in order to observe their influence. Moreover, deletion of the hormones from the cultures after as short a period as 12 h did not obviate their effects. There was no alteration of the kinetics of the response to PWM or an effect on the number of spontaneous PFC generated in vitro in the absence of PWM. In addition, there was no difference among men and women in response to either sex steroid, and within the female group, no variation was observed on different days of the menstrual cycle. These studies demonstrate direct immunoregulatory effects of specific sex steroids on human PBMC and support the idea that these hormones may have a role in the pathogenesis and treatment of some autoimmune disorders.