We have recently shown that the phorhol ester 12-O-tetradecanoyl-phorbol-13-acetate enhances the depolarization induced, calcium dependent release of [3H]dopamine from cultured brain neurons in the rat. In the present study the effects of 12-O-tetradecanoyl-phorbol-13-acetate on the kinetic parameters of depolarization induced calcium influx and on Ca2+ dependent neurotransmitter release and protein phosphorylation were investigated. Depolarization induced neurotransmitter release from the neurons occurs in two phases: an initial, last release and a subsequent slow release. At low extracellular Ca2+, 12-O-tetradecanoyl-phorbol-13-acetate enhanced the quantity of fast release and in addition, increased the rate constant of the slow release. These effects mimicked the effects of increasing the extracellular Ca+2. Various phorbol derivatives known to activate the Ca+2 activated phospholipid dependent protein kinase (protein kinase C) were also able to enhance the stimulated release of [3H]dopamine from the neurons. 12-O-tetradecanoyl-phorbol-13-acetate induced the incorporation of 32Pi into a protein with an apparent molecular weight of 45,000 daltons regardless of depolarization or of the presence of Ca2. In addition, 12-O-tetradecanoyl-phorbol-13-acetate induced in unstimulated neurons, Ca+2 dependent increase in the amount of32Pi incorporated into a 43,000 dalton protein and decrease in the amount incorporated into a 55,000 dalton protein. These changes mimicked the Ca+2 dependent changes in protein phosphorylation which occur upon stimulation of the neurons. Kinetic studies of depolarization induced Ca+2 uptake by the neurons indicated that 12-O-tetradecanoyl-phorbol-13-acetate enhanced the maximal influx of Ca+2 through the voltage sensitive Ca+2 channels by 40%. The results indicate that 12-O-tetradecanoyl-phorbol-13-acetatc acts primarily on the regulation of stimulated Ca24 entry into the cells. Consequently neurotransmitter release at suhmaximal extracellular [Ca+2] is enhanced.