We have found that maximum stimulation (greater than 10-fold) of kinase activity of a bovine brain preparation of calcium- and phospholipid-dependent protein kinase (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) occurs in the presence of phospholipid, but in the absence of added Ca2+. In effect, nM concentrations of TPA substitute for mM concentrations of added Ca2+, and the two agents are not synergistic. Biologically active analogs of TPA such as phorbol-12,13-dibutyrate (PDBu), 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA) or mezerein were also effective activators of PKC, as were the chemically unrelated tumor promoters teleocidin and aplysiatoxin, when tested at nM concentrations in the absence of added Ca2+. On the other hand, the biologically inactive compounds phorbol, 4-alpha-phorbol-12,13-didecanoate (4-alpha-PDD), HHPA-13,20-diacetate and 1,2-dihydro-20-deoxy-HHPA did not affect PKC activity in the absence or presence of Ca2+. Our results are consistent with a stereochemical model in which the hydrophilic domains of certain diterpenes, teleocidin and aplysiatoxin interact specifically with PKC apoenzyme, while their hydrophobic domains interact with phospholipid, thus forming an enzymatically active ternary complex.