Administration of a low dose of L-PAM (0.75 mg/kg) to mice bearing a large SC MOPC-315 tumor and extensive metastases led to the development of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells. Such drug-induced potentiation of antitumor immune responsiveness appeared by day 2 after chemotherapy, and it could not be further enhanced but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in depleting the cells known to have inhibitory activity (i.e., macrophages and metastatic tumor cells). To establish that L-PAM can lead to selective in situ abrogation of the inhibitory effectiveness of the splenic macrophages and metastatic tumor cells, we demonstrated that incubation of immunosuppressed tumor-bearer spleen cells with a low concentration of L-PAM in vitro also resulted in augmented antitumor immune potential that could not be further augmented by depletion of glass-adherent cells. L-PAM-mediated enhancement of the antitumor immune potential of immunosuppressed tumor bearer spleen cells was due at least in part to the effects of the drug on the splenic metastatic tumor cells. Isolated tumor cells treated with a low concentration of L-PAM were not only devoid of inhibitory activity for the primary in vitro antitumor immune response by normal spleen cells, but actually manifested a strong immunostimulatory capacity. Thus, L-PAM given at a low dose enhances the development of potent antitumor immunity which brings about the eradication of a large tumorigenic load that remains after the drug has been cleared from the circulation.