Major histocompatibility complex class II antigens are expressed on human T cells following activation, but their functional role remains obscure. We have investigated the effect of anti-class II monoclonal antibodies on T cell proliferation. Our results indicated that antibodies directed against either DR, DQ, or DP were able to decrease 3H-TdR uptake if the cells had been activated by interleukin-2 (IL-2). On the other hand, minimal inhibition resulted when phorbol dibutyrate, a phorbol ester, and ionomycin, a calcium ionophore, were used to activate cells. The specificity of the effect was demonstrated by the observation that anti-class I antibodies inhibited proliferation stimulated by IL-2 and phorbol dibutyrate and ionomycin equally well. Proliferation by the anti-class II monoclonal antibodies was inhibited regardless of whether the monoclonal antibodies were added at the initiation of culture or to actively proliferating cells, suggesting that an early event was not specifically targeted. Our findings are consistent with the interpretation that class II antigens are involved in the transmission of signals to activated T cells.