Pharmacological doses of estrogens such as 17-beta estradiol (17-beta E) and diethylstilbestrol (DES) suppress cell-mediated immunity in vivo. In this report, we investigated the direct in vitro effects of 17-beta E and its major metabolites on lymphocyte proliferation in response to the T cell lectin phytohemagglutinin (PHA). PHA-induced lymphocyte agglutination, an early event indicative of active, cytoskeletal-dependent membrane alterations, was monitored in conjunction with blastogenesis. Without exception, the effects of individual estrogen metabolites on the PHA-induced agglutination occurring within minutes were accompanied, at every concentration of compound, by equivalent effects on the blastogenic response of activated cells measured after several days. This observation suggested a role for estrogens in modulating lymphocyte activation at the cell surface rather than through cytosolic receptor-mediated events. As suggested by previous studies with quinone metabolites of benzene, the catechol estrogen metabolite 2-OH estrone (2-OH E) was significantly more potent than the parent compound at suppressing lymphocyte proliferation in vitro and in vivo.