Elicited guinea pig peritoneal macrophages (MPs) respond by an oxidative burst (OB) to a variety of membrane stimulants. Evidence has recently accumulated, indicating that phospholipase A2 activation resulting in unsaturated fatty acid liberation is a prerequisite for the induction of an OB by some stimulants. We examined the effect of inhibiting adenosylmethionine-dependent phospholipid methylation on the capacity of MPs to produce superoxide (O2-) in response to membrane stimulation. We found that preincubation of MPs with the transmethylation inhibitor, 3-deazaadenosine (DZAdo), totally eliminated the induction of an OB by concanavalin A, wheat germ agglutinin, and N-formyl-l-methionyl-l-leucyl-l-phenylalanine and partially blocked O2- production in response to NaF, phospholipase C, digitonin, the ionophore A23187, and phorbol myristate acetate (PMA). The PMA-elicited OB was the most resistant to inhibition by DZAdo. Homocysteine thiolactone enhanced the blocking effect of DZAdo. These findings suggest that stimulated O2- production by guinea pig peritoneal MPs requires enzymatic methylation of an unknown substrate, most likely a membrane phospholipid.