We studied both autophosphorylation and phosphotransferase activity of insulin receptors from abdominal skeletal muscles of nonobese subjects with non-insulin-dependent diabetes mellitus (NIDDM). Partially purified insulin receptors were labeled on their α-subunit with ¹²⁵I-labeled insulin by chemical cross-linking and on their β-subunit by autophosphorylation with 1000 μM ATP. Thereafter, phosphorylated insulin receptors were separated from total receptors with the anti-phosphotyrosine antibody. Thus, the percentage of phosphorylated receptors in total receptors revealed the autophosphorylation activity. Using this method, we studied the function of insulin receptors from muscle obtained by biopsy during surgery in 10 nonobese NIDDM and 8 control subjects. In diabetic subjects, insulin binding capacity from abdominal skeletal muscles was 69.4% of the control subjects. Furthermore, the percentage of phosphorylated insulin receptors stimulated by 8.3 nM insulin was significantly lower than the control subjects (mean ± SD, 29.0 ± 12.0 vs. 56.0 ± 7.4%, P < 0.01), and there was a significant inverse correlation between fasting plasma glucose levels and the percentage of phosphorylated receptors among diabetic subjects (r = 0.73, P < 0.025). Moreover, the insulin-stimulated kinase activity toward a synthetic peptide (Glu⁸⁰Tyr²⁰) was also impaired in diabetic subjects (28.5% of control). In summary, this is the first demonstration that the autophosphorylation step of insulin receptors from abdominal skeletal muscles is impaired in nonobese NIDDM subjects.