Little is known about the important cellular substrates for protein kinase C and their potential roles in mediating protein kinase C-dependent processes. We evaluated the protein kinase C phosphorylation sites in a major cellular substrate for the kinase, a protein of apparent Mr 80,000 in bovine and 60,000 in chicken tissues; we have recently determined the primary sequences of these proteins and tentatively named them the myristoylated alanine-rich C kinase substrates. The proteins were purified to apparent homogeneity from bovine and chicken brains, phosphorylated with protein kinase C, digested with trypsin, and the phosphopeptides purified and sequenced. Four distinct phosphopeptides were identified from both the bovine and chicken proteins. Two of the phosphorylated serines were contained in the repeated motif FSFKK, one in the sequence LSGF, and one in the sequence SFK. All four sites were contained within a basic domain of 25 amino acids which was identical in the chicken and bovine proteins. All of the sites phosphorylated in the cell-free system appeared to be phosphorylated in intact cells; an additional site may have been present in the proteins from intact cells. The identity of the phosphorylation site domains from two proteins of overall 65% amino acid sequence identity suggests a potential role for this domain in the physiological function of the myristoylated alanine-rich C kinase substrate proteins.