The involvement of arachidonic acid and arachidonic acid metabolites in the control of oxytocin secretion by ovine corpus luteum was investigated, using slices of luteal tissue incubated in vitro. Oxytocin was secreted at steady rates by luteal slices, during 60-min incubations (315.0 ± 45.3 pg/mg-h). The secretion of oxytocin was stimulated by arachidonic acid, phospholipase A2 (PLA2), and phospholipase C (PLC) in a dosedependent manner. The highest doses of arachidonic acid, PLA2) and PLC used stimulated oxytocin secretion by 145.8 ± 23.0% (P < 0.01; n = 6), 331.5 ± 42.4% (P < 0.02; n = 4), and 955.5 ± 278.6% (P < 0.01; n = 4), respectively. Oxytocin secretion by luteal slices was not affected by either prostaglandin F2α (PGF2α) or PGE2 over a concentration range from 3–3000 nM. Furthermore, inhibitors of the cyclo-oxygenase pathway of arachidonic acid metabolism did not consistently affect arachidonic acid and PLA2-stimulated oxytocin secretion. Nordihydroguaiaretic acid, which inhibits 5-lipoxygenase, however, totally abolished arachidonic acid- and reduced PLA2-stimulated oxytocin secretion. The presence of CoCl2 in the incubation medium also significantly reduced basal and PLA2- and PLC-stimulated oxytocin secretion [P < 0.05 (n = 5), P < 0.05 (n = 5), and P < 0.01 (n = 6), respectively]. We have shown that oxytocin secretion from slices of ovine corpus luteum incubated in vitro is stimulated by exogenous and endogenously released arachidonic acid. The data show that PGF2α and PGE2 do not have a role in luteal oxytocin secretion in vitro and PG formation does not appear to be involved in the stimulation of oxytocin secretion elicited by arachidonic acid or PLA2. Arachidonic acid may have its effect via the lipoxygenase pathway.