Peripheral blood polymorphonuclear neutrophils (PMN) can significantly inhibit lymphokine-activated killer- (LAK) mediated cytotoxicity when added to a cytotoxicity assay of IL-2-activated PBL and target cells. The inhibition by resting PMN is resistant to blocking with catalase and superoxide dismutase, suggesting that reactive oxygen species are not involved. The addition of TNF greatly enhanced the PMN-mediated inhibition of LAK effector functions. This TNF-enhanced inhibition is reversed by catalase, but not by superoxide dismutase, implicating hydrogen peroxide in the augmented inhibition. Separation of PMN from effector cells and target cells totally abrogates the inhibition by both resting PMN and TNF-treated PMN. Formalin-fixed PMN, heat-treated PMN, PMN lysates, and PMN membrane all fail to mediate any inhibition of LAK. These results suggest that contact with intact viable PMN is needed for inducing LAK inhibition. However, pretreatment of LAK cells with PMN also decreases their cytotoxicity in subsequent chromium release assays. PMN can also inhibit NK cytotoxicity of fresh PBL. However, NK activity is much less sensitive to inhibition by resting PMN than is LAK. TNF also augments PMN inhibition of NK, and there is no significant difference between LAK and NK in sensitivity to the TNF-enhanced inhibition. Our results indicate that PMN can significantly influence the destruction of tumor targets by LAK and NK, and suggest that approaches to circumvent such regulation may be important in the outcome of immunotherapies with IL-2 and LAK cells.