We have utilized 4-hydroperoxycyclophosphamide (4-HP-CY) as a probe for the immunomodulatory activity of the metabolites of cyclophosphamide (CY) since 4-HP-CY hydrolyzes spontaneously in aqueous solution to the same metabolites as those formed after in vivo conversion of CY by microsomal enzymes. Exposure of immunosuppressed MOPC-315 tumor bearer spleens to a low concentration of 4-HP-CY (0.1-3.0 micron) resulted in augmented antitumor immune potential. The level of antitumor immune potential exhibited by 4-HP-CY-treated tumor bearer spleen cells was not further augmented but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in removing the cells known to have immunosuppressive activity (i.e. metastatic tumor cells and macrophages) from the spleen of untreated MOPC-315 tumor-bearing mice. In fac, 4-HP-CY was superior to depletion of glass-adherent cells in augmenting the antitumor immune potential of immunosuppressed tumor bearer spleen cells. When cells from the primary tumor nodule were incubated with a low concentration of 4-HP-CY which only marginally inhibited their proliferation, the drug completely abolished the suppressive activity of the cells for in vitro generation of antitumor cytotoxicity by normal spleen cells. Moreover, a high level of antitumor cytotoxicity developed when normal spleen cells were cultured in vitro with 4-HP-CY-treated tumor cells at a wide range of ratios of spleen cells to tumor cells. Thus, in the MOPC-315 tumor model, metabolites of CY eliminate the inhibitory effectiveness of splenic suppressor cells and induce the appearance of immunopotentiating activity. The results obtained with 4-HP-CY in vitro provide support for the hypothesis that low-dose CY therapy of mice bearing a large MOPC-315 tumor leads to the appearance of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells as a result of the in situ immunomodulatory effect of the drug on cells in the spleen.