The serine/threonine kinase Akt (PKB/Rac) has been implicated as playing a role in the insulin-signaling pathway to glucose transport. Little is known regarding the regulation of Akt kinase activity in insulin-sensitive tissues, such as skeletal muscle, or whether this regulation is altered in insulin-resistant states such as NIDDM. We examined the effect of insulin on Akt kinase activity in skeletal muscle from six NIDDM patients and six healthy subjects. Whole-body insulin sensitivity, assessed by the euglycemic-hyperinsulinemic clamp, was significantly lower in NIDDM subjects (P < 0.001), and this was accompanied by impaired in vitro insulin-stimulated glucose transport in skeletal muscle. In both groups, insulin induced a significant increase in Akt kinase activity, but the response to maximal insulin (60 nmol/l) was markedly reduced in skeletal muscle from NIDDM subjects (66% of control levels, P < 0.01). Impaired Akt kinase activity was not accompanied by decreased protein expression of Akt. Instead, a trend toward increased Akt expression was noted in skeletal muscle from NIDDM subjects (P < 0.1). These parallel defects in insulin-stimulated Akt kinase activity and glucose transport in diabetic skeletal muscle suggest that reduced Akt kinase activity may play a role in the development of insulin resistance in NIDDM.