This study was performed to investigate whether 85 kDa cytosolic phospholipase A2 (cPLA2) functions in eicosanoid generation in rat corpora lutea (CL) using its specific inhibitor, arachidonyl trifluoromethyl ketone (ATK). In both immature and adult pseudopregnant rats, PLA2 activity in the cytosol of CL, measured by the liposome-vesicle assay, increased from day 6 of pseudopregnancy (PSP6) to PSP12. 10 microM ATK potently inhibited all of these activities to 10-20% and the rate of the inhibition by ATK was much higher on PSP12. ATK also reduced arachidonic acid (AA) release from luteal cells of PSP12 prelabelled with 3H-AA. Furthermore, the production of prostaglandin E2 by cultured luteal cells was mostly suppressed by the drug. These results suggest the augmentation of cPLA2 activity with the luteal age of pseudopregnant rats and its principal role in eicosanoid generation in CL.