Monocytes, recovered by centrifugal elutriation, effectively inhibit functions of human NK cells in vitro. Histamine, acting via monocyte H2-type histamine receptors, abrogates the inhibitory signal. The aim of this study was to define the histamine-reversible mechanism by which monocytes inhibit NK cells with special reference to the respiratory burst activity of monocytes. Monocytes recovered from patients with chronic granulomatous disease did not suppress NK cell function, indicating the requirement of intact nicotinamide-adenine dinucleotide phosphate (NAPDH) oxidase activity of monocytes to inhibit NK cells. Furthermore, catalase, a scavenger of hydrogen peroxide (H2O2), was found to potently reverse the monocyte-induced inhibition of NK cell function; on the other hand, superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, the hypochlorous acid scavenger taurin, or the nitric oxide synthetase inhibitor NG-monomethyl-L-arginine (L-NMMA) did not affect the monocyte-derived, suppressive signal. H2O2, at micromolar concentrations, reconstituted the inhibitory effects of monocytes on NK cell function. Histamine had no scavenger activity but effectively suppressed the generation of H2O2 in isolated monocytes. This effect of histamine was transduced by H2-type histamine receptors. We conclude that the histamine-reversible, inhibitory effect of elutriated monocytes on NK cells is dependent on the formation of reactive oxygen metabolites by monocytes and that H2O2 is a pivotal mediator of the suppressive signal.