Inhibitors of arachidonic acid oxygenation can inhibit polymorphonuclear leukocyte lysosomal enzyme release, aggregation, and generation of activated oxygen species, as stimulated by N-formylated chemotactic peptides. Guinea Guinea pig peritoneal leukocytes were prelabeled with [3H]arachidonic acid, and 6-keto-prostaglandin F1 alpha, thromboxane B2, prostaglandin E2, prostaglandin F2 alpha, and 5-monohydroxyeicosatetraenoic acid were identified as products produced by these cells. Both Ionophore A23187 and the N-formylated peptide, N-formylmethionylphenylalanine, significantly stimulated release of these products, with the exception of a lack of effect of N-formylmethionylphenylalanine on 6-keto-prostaglandin F1 alpha. This metabolite was found to be produced by contaminating mononuclear cells unresponsive to N-formylmethionylphenylalanine stimulation when cell preparations purified to 96 leads to 99% polymorphonuclear cells were studied. The ability of N-formylmethionylphenylalanine to stimulate arachidonic acid metabolism of polymorphonuclear leukocytes was specifically antagonized by a competitive blocker of the formylated peptide receptor, t-butoxy-carbonyl-leucyl-phenylalanyl-leucyl-phenylalanine. The results indicate that metabolism of arachidonic acid induced by N-formylmethionylphneylalanine could play a role in the responses of polymorphonuclear leukocytes to this peptide.