Phorbol esters stimulate the phosphorylation of the insulin receptor on discrete serine and threonine residues in intact cells. Phosphorylation of the insulin receptor cytoplasmic domain on serine, threonine, and tyrosine residues regulates receptor tyrosine kinase activity and signaling. In these studies, we demonstrate that phorbol ester treatment of intact COS-1 cells transiently expressing the human insulin receptor stimulates phosphorylation of serine 1327 within the carboxyl-terminal tail of the insulin receptor beta subunit. Phosphopeptide maps of wild-type (Ser1327) and mutant (Ala1327) human insulin receptors revealed the absence of a single phosphopeptide in the Ala1327 receptors when compared with wild-type receptors from phorbol ester-treated cells. Phosphoamino acid analysis revealed phosphoserine within the phosphopeptide from wild-type receptors that is absent in the Ala1327 receptor. The synthetic peptide 1327S (KRSYEEHIPYTHMNGGKK) corresponding to amino acids 1325-1342 of the human insulin receptor is phosphorylated on serine by protein kinase C. After digestion with trypsin, the phosphorylated synthetic peptide comigrated with the serine-phosphorylated peptide isolated from wild-type insulin receptors that was absent from the Ala1327 mutant. Ser1327 is proximal to autophosphorylation sites Tyr1328 and Tyr1334. The potential effects of serine phosphorylation at position 1327 on subsequent phosphorylation of these tyrosines by the insulin receptor kinase were examined using synthetic peptides. The chemically modified peptide 1327S(P) was synthesized with the stoichiometric addition of phosphate to the side chain hydroxyl of a serine corresponding to position 1327 of the insulin receptor. Kinetic analysis revealed that the addition of phosphate to the serine improved substrate recognition by the insulin receptor tyrosine kinase almost 2-fold. The average Km was 1.44 mM for the peptide 1327S(P) versus 2.64 mM for peptide 1327S. However, when compared with the unphosphorylated control peptide, 1327S, the serine-phosphorylated peptide 1327S(P) also reduced the Vmax of the insulin receptor tyrosine kinase 53%. Radiosequence analysis revealed that the chemical addition of phosphate to the serine in peptide 1327S(P) inhibited insulin receptor-catalyzed phosphorylation of the tyrosine on 1327S(P) corresponding to Tyr1334 but not of the tyrosine corresponding to Tyr1328. These data suggest that the juxtaposition of a serine phosphorylation site adjacent to receptor tyrosine phosphorylation sites provides the potential for regulation of insulin receptor autophosphorylation and signaling through its carboxyl-terminal tail.