We used in vivo and in vitro assays to determine whether suppressor cells are generated in mice rendered unresponsive to tumor-specific antigens by intravenous (i.v.) injection of non-replicating tumor cells. The results show that a single i.v. injection of 2 x 10(7) irradiated P815 tumor cells resulted in the induction of a state of specific unresponsiveness to tumor-associated antigens, as revealed by the inability of the treated mice to achieve immunologically-mediated regression of an intradermal P815 tumor containing C. parvum, or to generate effector T cells capable of causing rejection of a P815 tumor in T-cell-deficient (T x B) test recipients. Failure to respond to tumor antigens was associated with the presence in spleen of CD4+ T cells capable, on passive transfer, of suppressing adoptive T-cell-mediated tumor regression in T x B recipients. However, the same CD4+ suppressor cells failed to inhibit the generation of tumor-specific cytotoxic T lymphocytes (CTL) in vitro. On the contrary, spleen cells from mice made unresponsive by i.v. injection of tumor cells were primed to generate CTL in response to tumor antigens in vitro. Taken together, our results suggest that unresponsiveness induced by i.v. injection of tumor antigens is an active process mediated, at least in part, by CD4+ T suppressor cells, and that these cells coexist in the spleen with antigen-primed effector T cells with a capacity to generate tumor-specific CTL when released from suppression in vitro.