We examined the effects of EGTA and verapamil on phorbol ester-and forskolin-stimulated LH releases and LHβ subunit mRNA levels in order to verify the role of extracellular Ca++ on PKC- or cAMP-induced increases in LH release and LHβ subunit mRNA levels in cultured anterior pituitary cells of rat. Forskolin-stimulated LHβ subunit mRNA levels as well as LH release were all suppressed by prevention of Ca++ mobilization from extracellular environment, after the treatment of EGTA as a Ca++ chelator or verapamil as a Ca++ channel blocker. PMA-stimulated LHβ subunit mRNA levels were also suppressed by the treatment of EGTA and verapamil, while PMA-induced LH release was not affected. From the present study, it is, therefore, suggested that PKC activation and cAMP elevation all stimulate LHβ subunit mRNA levels and these are extracellular Ca++-dependent. However, LH releases by PKC activation and cAMP increase seem to be different each other. LH release by PKC activation is thought to be independent of extracellular Ca++. On the other hand, cAMP stimulated-LH release is thought to be dependent on the entry of extracellular Ca++.