In a previous report we described Ser(1275) and Ser(1309) as autophosphorylation sites of the human insulin receptor (IR) tyrosine kinase (TK) in vitro. The question remained whether the observed phosphorylation was exclusive for the in vitro activated receptor or a more general, mechanism of the activated receptor in situ. In this study, we determined the intrinsic activity of the IR to phosphorylate both serine residues in intact cells, For this purpose CHO-09 and NIH-3T3 derived cell-lines expressing the human IR were metabolically labelled with [P-32]orthophosphate, followed by hormone stimulation of the receptor. The IR was isolated by immunoprecipitation and SDS-PAGE and subsequently analysed for serine phosphorylation by phosphopeptide mapping of HPLC-purified tryptic phosphopeptides. Activation of the IR in the intact cell appeared to result in phosphate incorporation into Ser(1275) and Ser(1309), providing strong evidence that both serine residues are phosphorylation sites of the activated receptor in intact cells,