When RAW264.7 murine macrophages were incubated with cholera toxin or pertussis toxin, prostaglandin E2 (PGE2) synthesis was enhanced markedly. Cholera toxin and pertussis toxin added together synergistically stimulated PGE2 synthesis. Cholera toxin and pertussis toxin also stimulated cyclic AMP (cAMP) accumulation. However, PGE2 synthesis was independent of increases in cAMP, as neither forskolin nor isoproterenol, which increased cAMP accumulation, nor dibutyryl-cAMP had any effect on PGE2 synthesis. In intact cells, cholera toxin and pertussis toxin stimulated phospholipase A2 to enhance metabolism of phosphatidylinositol to lysophosphatidylinositol and glycerophosphoinositol, with time courses similar to their stimulation of PGE2 synthesis. Cholera toxin catalyzed ADP-ribosylation of proteins of Mr 45,000 and 49,000 in intact cells, whereas an additional substrate of Mr 41,000 was observed in vitro. Preincubation of intact cells with pertussis toxin blocked subsequent in vitro labeling of the Mr 41,000 protein by cholera toxin, suggesting that the same protein was ADP-ribosylated by both toxins. Western blot analysis using specific antisera against Gi, Go and Gs revealed that the Mr 41,000 substrate was bound by the anti-Gi and anti-Go but not anti-Gs. The present data suggest that guanine nucleotide binding regulatory proteins are involved in the regulation of arachidonic acid metabolism to PGE2 in RAW264.7 cells. Furthermore, the possibility is raised that phospholipase A2 is regulated by both stimulatory and inhibitory guanine nucleotide binding proteins.