Abstract Periods of overinsulinization with low blood glucose levels are a recognized feature of intensive insulin injection therapy. The relationship of these features to insulin insensitivity is controversial, and the mechanisms underlying any such changes are unclear. Normal rats have therefore been overinsulinized for 6 weeks before measurement of in vivo insulin sensitivity by the glucose clamp technique. Skeletal muscle glycogen synthase and pyruvate dehydrogenase activities were measured at the end of the clamp. Sensitivity to insulin as measured by the glucose clamp technique at euglycemic levels was decreased in the insulin overtreated animals (glucose requirements, 108 ± 2 μmol/min/kg v 170 ± 10 μmol/min/kg, P < 0.001). Total skeletal muscle glycogen synthase activity was increased in the experimental group (2.83 ± 0.12 v 1.96 ± 0.14 U/g wet weight, P < 0.001), and as a result the active fraction was higher at the end of the clamp (0.79 ± 0.04 v 0.66 ± 0.04 U/g wet weight, P < 0.05). Skeletal muscle glycogen content was consistent with the glycogen synthase activity. Pyruvate dehydrogenase in the same tissue showed increased activation prior to the clamp (6.6 ± 0.6 v 4.7 ± 0.6%, P < 0.05), but neither active nor total activity was abnormal at the end of the clamp. Thus overinsulinization decreases insulin sensitivity, but this change cannot be accounted for by changes in the activities of these two key enzymes of glucose disposal.