Protein kinase C is specifically regulated by diacylglycerol and the amino phospholipid, phosphatidylserine. The molecular basis for the phosphatidylserine specificity was recently proposed to arise from the presence of a putative phosphatidylserine binding motif, FXFXLKXXXKXR, localized in the C2 domain of protein kinase C (Igarashi, K., Kaneda, M., Yamaji, A., Saido, T. C., Kikkawa, U., Ono, U., Inoue, K., and Umeda, M. (1995) J. Biol. Chem. 270, 29075-29078). To determine whether this motif mediates the interaction of protein kinase C with phosphatidylserine, the carboxyl-terminal basic residues were mutated to Ala in protein kinase C betaII (K236A and R238A), and the phosphatidylserine regulation of the mutant enzyme was examined. Membrane binding and activity measurements revealed that the phosphatidylserine regulation for the mutant protein was indistinguishable from that of wild-type protein kinase C. Specifically, neither the apparent membrane affinity for phosphatidylserine-containing membranes in the presence or absence of diacylglycerol nor the phosphatidylserine-dependence for activation was affected by removal of the conserved basic residues at the carboxyl terminus of the consensus sequence. In addition, a synthetic peptide corresponding to the amino terminus of the consensus sequence (FTFNVK) had no effect on the concentration of phosphatidylserine resulting in half-maximal activation of protein kinase C. These results reveal that the carboxyl-terminal basic residues in the consensus motif FXFXLKXXXKXR are not responsible for the phosphatidylserine selectivity of protein kinase C and that, furthermore, the region of the C2 domain containing this motif is not involved in the membrane binding of protein kinase C.