Treatment of adult mice with high doses of the immunosuppressive drug cyclophosphamide (CY) induces transient splenic natural suppressor (NS) cell activity mediated largely by cells bearing the MAC-1+ cell-surface marker. Here we show that culture supernatants from mixed lymphocyte reactions (MLR) suppressed by MAC-1+ NS cells exhibit decreased IL-2 and IL-4 activity in bioassays for these lymphokines. However, inhibition of MLR was maximal whether the regulatory cells were added at initiation of culture or 24 hr postinitiation, suggesting that inhibition of lymphokine synthesis is not likely to be the reason for diminished lymphocyte proliferation, since these particular lymphokine genes are known to be transcribed and expressed during the first 12 hr of culture. Furthermore, flow cytofluorometric analysis demonstrated that the presence of MAC-1+ NS cells did not alter the percentage of lymphokine-producing CD4+ T cells in MLR. IL-2 receptor (p55) expression was also normal in suppressed MLR. The addition of exogenous IL-2 and/or IL-4 to MLR failed to reverse the inhibitory effect of MAC-1+ NS cells on lymphocyte proliferation, indicating that these regulatory cells block the utilization of these lymphokines in MLR. The inhibitory effect of MAC-1+ NS cells on lymphocyte proliferation in MLR is dependent on interferon-gamma, since NS activity was dramatically decreased in the presence of neutralizing antibodies to interferon-gamma. MAC-1+ NS cell-induced suppression of MLR was also diminished in the presence of indomethacin, suggesting that prostaglandins play a role in this NS system.