Plasma membrane samples prepared from corpora lutea (CL) of control and prostaglandin F2 alpha-treated rats were incubated with radiolabeled phosphatidylcholine for 90 min at 40 C with 1 mM CaCl2 to test for the presence of phospholipase A2 activity. At the end of the incubation period, labeled arachidonic acid cleaved from the 2 position of the phospholipid moiety had accumulated to 10.7 +/- 2.4% (mean +/- SE) of the initially added radioactivity in the prostaglandin F2 alpha-treated samples, with 3.7 +/- 1.2% appearing in the controls. Arachidonic acid production was inhibited by calcium chelation and by the phospholipase A2 inhibitor bromophenacyl bromide, indicating heightened activity of phospholipase A2 in CL plasma membranes undergoing regression. Unexpectedly, radiolabeled lysophosphatidylcholine was produced in regressed membrane samples at a similar rate, suggesting the induction of phospholipase A1 activity as well. To determine if the membrane rigidification that occurs with regressed membrane samples under the same incubation conditions is caused by the hydrolysis products of phospholipase activity, fluorescence polarization experiments with the probe trans-parinaric acid were conducted. Washing of incubated membrane samples with fatty acid-free BSA, which selectively removes free fatty acids and lysophosphatides from the bilayer, resulted in a restoration of the fluidity to levels recorded at the onset of the incubation. These results suggest that the previously described decreases in CL plasma membrane fluidity and hCG binding in vitro during luteolysis are caused by a synergistic effect of calcium ion and hydrolysis products of phospholipase A activity.