We previously reported that prostaglandin F2 alpha (PGF2 alpha) activates phosphatidylcholine-hydrolyzing phospholipase D independently from the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells, and reported that pertussis toxin-sensitive GTP-binding protein (G-protein) is involved in the PGF2 alpha-induced phospholipase D activation. In this study, we examined the effect of retinoic acid (RA) on the phospholipase D activity stimulated by PGF2 alpha in these cells. The pretreatment of RA markedly inhibited the formation of choline induced by PGF2 alpha (10 microM) in a dose-dependent manner in the range between 1 nM and 0.1 microM. This inhibitory effect of RA was dependent on the time of pretreatment up to 8 h. However, RA had little effect on the choline formation induced by NaF, a G-protein activator, or 12-O-tetradecanoylphorbol-13-acetate, an activator of PKC. These results strongly suggest that RA suppresses the phospholipase D activated by PGF2 alpha in osteoblast-like cells and that the effect of RA is exerted at the point between PGF2 alpha receptor and G-protein.