In rat skeletal muscle, calcitriol, the hormonal form of vitamin D3, rapidly stimulates the biphasic formation of diacylglycerol (DAG), the second phase being independent of phosphoinositide hydrolysis driven by phospholipase C. In this work we showed that the effect of calcitriol on the second phase of DAG formation was totally inhibited in the absence of extracellular Ca2+ and by the Ca2+-channel blockers nifedipine and verapamil, whereas the Ca2+ ionophore A23184, similar to calcitriol, increased DAG formation by 100%. GTPgammaS, which activates G protein-mediated signals, mimicked the effects of the hormone while GDPbetaS, an inhibitor of G proteins, suppressed calcitriol-induced DAG formation. To elucidate the metabolic pathway of the late phase of DAG production, we examined the contribution of phospholipase D (PLD), which acts on phosphatidylcholine (PC) generating phosphatidic acid that is converted to DAG by a phosphatidate phosphohydrolase. In [3H]arachidonate-labeled muscle, calcitriol increased [3H]phosphatidylethanol (PEt) formation in the presence of ethanol, a reaction specific for PLD. The effects of the hormone were time- and dose-dependent with maximum PEt levels achieved at 10(-9) M. The phorbol ester TPA also stimulated PEt formation. The combination of calcitriol and TPA was more effective than either compound alone. In rat muscle, calcitriol increased PKC activity in a time-dependent fashion. Bisindolymaleimide, a selective inhibitor of the enzyme, completely suppressed TPA-induced PEt and attenuated the effects of the hormone. These results provide the first evidence concerning calcitriol stimulation of the hydrolysis of PC in a mammalian tissue through a phospholipase D catalyzed mechanism involving Ca2+, protein kinase C, and G proteins.