The release of luteal oxytocin during spontaneous and prostaglandin-induced luteolysis was investigated in cows. A continuous-flow microdialysis system was used in 11 cows to collect dialysates of the luteal extracellular space between Days 12 and 24 postestrus. Seven cows were untreated and were expected to exhibit spontaneous luteolysis during sampling, whereas 4 cows received prostaglandin F_2α (PGF_2α) systemically between Days 13 and 15 to induce luteolysis during sampling. Oxytocin was detectable in the dialysate of all cows before Day 16 postestrus and occurred as 2 or 3 discrete pulses per 12-h sampling period. For non-PGF_2α-treated cows, dialysate oxytocin content began to decline spontaneously on Day 15 postestrus and was undetectable by Day 17 postestrus. Oxytocin decay curves preceded onset of serum progesterone decline by at least 72 h and were not related temporally with onset of progesterone decline within cow. Exogenous PGF_2α (25 mg, i.m.) produced a 10-fold increase in dialysate oxytocin within 1 h (1.9 ± 0.3 pg/ml to 20.8 ± 3.0 pg/ml; P < 0.01). Dialysate oxytocin then declined to pretreatment concentrations within 2 h and was undetectable within 8 h posttreatment. A second PGF_2α injection given 20 h after the first did not result in a measurable increase in dialysate oxytocin, probably because luteolysis was underway. Although robust luteal oxytocin release was observed after treatment with a pharmacological dose of PGF_2α, the lack of detectable oxytocin secretion during spontaneous luteolysis suggests that the contribution of luteal oxytocin in the cow may be less than that proposed for the ewe.