Autophosphorylation sites of the human insulin receptor were identified by microsequence analysis of 19 discrete tryptic [32P]phosphopeptides, purified from the autophosphorylated cytoplasmic kinase domain (CKD). Seventeen phosphopeptides were generated by cleavage at Arg and/or Lys, but two phosphopeptides were generated reproducibly by anomalous cleavages. Two new sites were identified in the juxtamembrane region of the intact insulin receptor beta-subunit (the amino terminus of the CKD), including phosphotyrosines 965 and 972. Three sites in the central region, including phosphotyrosines 1158, 1162, and 1163, were identified from six phosphopeptides; tyrosine at 1158 was the least phosphorylated. Monophosphopeptides contained phosphotyrosine at either residue 1158 or 1163, but not at 1162. Bisphosphorylation included phosphotyrosines only at 1162 and 1163. The two autophosphorylation sites near the carboxy terminus were found in seven phosphopeptides, including phosphotyrosines at 1328 and 1334. When mapped by reverse-phase high-performance liquid chromatography, these phosphopeptides eluted in the order central domain, first; carboxy-terminal region, second; and juxtamembrane (amino-terminal) domain, last.