The activity of phospholipase A2 was measured in microsomes prepared from ovaries of superovulated pseudopregnant rats during spontaneous and prostaglandin F2 alpha-induced regression and during regression in pregnant rats. Microsome samples were incubated at 40 C for 90 min in Tris buffer (pH 8.3) with 1.0 mM CaCl2 added. The substrate, radiolabeled phosphatidylcholine, was incorporated into liposomes. During spontaneous regression, there was a significant 2- to 4-fold increase in phospholipase A2 activity, when compared with levels at mid-pseudopregnancy (days 8-9). This elevation was correlated with a significant decrease in plasma progesterone concentration. On day 6 or 7 of pseudopregnancy, treatment of rats with luteolytic doses of prostaglandin F2 alpha also caused a significant increase in phospholipase A2 activity, which remained elevated throughout the 72-h sampling period. In pregnant rats there was a small but significant rise in phospholipase A2 activity after parturition. These results indicate that the activity of phospholipase A2 increases during luteal regression in pregnant and pseudopregnant rats and that it could be involved in the mechanism that causes the loss in progesterone secretion.