The effect of prostaglandin F2 alpha (PGF2 alpha) on superoxide radical production by macrophages was studied in pseudopregnant rats. Peritoneal macrophages prepared on day 7 or 13 of pseudopregnancy (psp) were incubated with various doses of PGF2 alpha for 90 min, and the production of superoxide radical was measured by the cytochrome C reduction method. PGF2 alpha significantly stimulated superoxide radical production by macrophages on day 13 of psp, but not on day 7 of psp. The pretreatment of macrophages with an inhibitor of protein kinase C (H7), Ca2+ channel blocker (Verapamil), Ca2+ chelators (EGTA, BAPTA), and an inhibitor of GTP-binding protein (pertussis toxin) prevented the stimulatory effects of PGF2 alpha on superoxide radical production. In conclusion, PGF2 alpha stimulated superoxide radical production by macrophages through the intracellular signal transduction pathway including activation of protein kinase C through the GTP-binding protein and Ca2+ influx, which would play important roles in the luteolytic process in psp rats.