Insulin stimulated autophosphorylation of the P-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the P-subunit: the regulatory region containing Tyr-1 146, Tyr-1 150, and Tyr-1 15 1, and the C-terminus containing Tyr-13 16 and Tyr-1322. In the presence of antiphosphotyrosine antibody (a-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that a-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1 15 1. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of a-PY, which contained Tyr(P)-l146 and either Tyr(P)-l150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the P-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the P-subunit was mainly (>SO%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the 0-subunit (White et al.:Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.