In cultures of rat granulosa cells, luteinizing hormone-releasing hormone (LHRH) increases32P incorporation into both phosphatidylinositol (PI) and phosphatidic acid (PA). After 20 min, the level of radioactivity was three- to four-fold (p < 0.01) above control in the PI and PA fractions, respectively. The stimulatory effect of LHRH on32P incorporation was limited to PI and PA. Similar to the effects of LHRH, a rapid and marked increase of32P incorporation into both PI and PA is observed upon addition of prostaglandin F2α(PGF2α) (10−5 M) to rat granulosa cells. Incorporation of radioactivity into PA was already increased (p < 0.05) by 2 min following PGF2αaddition, while the increase in32P-labeled PI became significant (p < 0.01) by 5 min. In contrast to PGF2α, the labeling of PI and PA following the addition of PGE2(10−5 M) was not significantly different from control levels during the entire 10 min of incubation. The sensitivity of the increased PA–PI labeling induced by LHRH and PGF2αis compared in another experiment. After 20 min incubation 10−6 MLHRH increased PI and PA labeling by six-and four-fold, respectively. Although the effect of PGF2αis less than that of LHRH, 10−5 MPGF2αsignificantly (p < 0.01) increased PI and PA labeling by three- and two-fold, respectively. By contrast, 10−6 MPGE2failed to affect32P incorporation into the various phospholipid fractions, but a small enhancement (p < 0.05) of PI and PA labeling was observed only at 10−5 MPGE2. These data further support the hypothesis that LHRH and PGF2αshare a common mechanism in ovarian cells involving phospholipid metabolism.