We previously reported that prostaglandin F 2 α (PGF 2 α ) induces Ca 2 + influx from the extracellular space via protein tyrosine kinase in osteoblast-like MC3T3-E1 cells and that PGF 2 α stimulates phosphatidylcholine-hydrolyzing phospholipase D in these cells (6, 12). In this study, we examined the relationship between the tyrosine kinase-regulated Ca 2 + influx by PGF 2 α and the activation of phospholipase D in MC3T3-E1 cells. The depletion of extracellular Ca 2 + by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) markedly reduced the PGF 2 α -induced formation of choline. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on choline formation, significantly suppressed the formation of choline induced by PGF 2 α in a dose-dependent manner. Tyrphostin, an inhibitor of protein tyrosine kinases chemically distinct from genistein, also suppressed the PGF 2 α -induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, significantly enhanced the PGF 2 α -induced formation of choline. These results strongly suggest that the phospholipase D activation by PGF 2 α is dependent on extracellular Ca 2 + in osteoblast-like cells and that protein tyrosine kinase is involved in the activation of phospholipase D.