The G protein alpha subunit Gz alpha is a substrate for phosphorylation by protein kinase C. The phosphorylation has been documented both in human platelets and in vitro and is characterized by a high degree of selectivity in relation to other G protein alpha subunits. We have demonstrated previously by phosphoamino acid analysis and CNBr peptide mapping that phosphorylation occurs at a serine residue(s) within the NH2-terminal 53 residues of Gz alpha. In this study, we have examined the site of phosphorylation using site-directed mutagenesis. Gz alpha variants containing selected substitutions of alanine for serine residues were expressed in human kidney 293 cells, and the ability of each to be phosphorylated in response to phorbol 12-myristate 13-acetate was examined. A focus was placed on Ser25 and Ser27, the 2 serine residues within a sequence of Gz alpha used to obtain a phosphorylation-sensitive antibody. The results demonstrate that Ser27 is the primary site of phosphorylation. Conversion of Ser27 to an alanine resulted in a 65% decrease in incorporation of [32P] phosphate; conversion of Ser25 had no effect. Conversion of Ser16, which like Ser25 and Ser27 conforms to a consensus site for protein kinase C, resulted in a modest (15%) decrease. The conversion of both Ser16 and Ser27 resulted in an 80% suppression of incorporation. In addition to these results, we have extended studies of the subunit and kinase selectivity of phosphorylation in platelets. We show here that under conditions promoting phorbol 12-myristate 13-acetate-stimulated phosphorylation of Gz alpha in permeabilized platelets, Gq alpha is not phosphorylated. Moreover, Gi alpha, Gz alpha, and Gq alpha were not phosphorylated in response to analogues of cAMP or cGMP. Thus, only Gz alpha is phosphorylated in platelets and only in response to activation of protein kinase C.