Activities of Akt1, Akt2, and Akt3 kinases and glucose uptake in hindlimb muscles of the rat in vivo were investigated. The rats were studied either after intravenous injection of 0.1 U of insulin or during exercise induced by stimulating calf muscles electrically at 1 contraction/s. Akt kinases were immunoprecipitated from supernatants of muscle homogenates. Glucose uptake by muscles in vivo was assessed by cellular accumulation of 2-deoxy-D-[1, 2-3H(N)]glucose. Administration of insulin resulted in rapid activation of Akt1 kinase, with peak activity observed 5 min after insulin injection. Soleus muscle, a slow-twitch muscle, and plantaris muscle, a fast-twitch muscle, differed in their content of Akt1 kinase and in their response to insulin. Soleus muscle exhibited a 105% higher abundance of Akt1 kinase, a 101% higher insulin-stimulated activity of Akt1 kinase, and 83% higher insulin-stimulated 2-deoxyglucose uptake compared with plantaris muscle. Additionally, insulin administration increased the activities of Akt1, Akt2, and Akt3 kinases in calf muscles and caused a sevenfold augmentation in 2-deoxyglucose uptake by these muscles. In contrast, the exercised calf muscles exhibited an increase in Akt1 kinase activity at 5, 15, and 25 min of exercise but no change in activities of Akt2 and Akt3 isoforms, and the 2-deoxyglucose uptake by calf muscles exercised for 25 min was 11-fold higher compared with muscles of resting rats. The data demonstrate that 1) there is a close, direct correlation between the magnitude of insulin-stimulated activity of Akt1 kinase and the level of glucose uptake in muscles with different fiber populations, 2) insulin activates three isoforms of Akt kinase in skeletal muscle, and 3) exercise in vivo is associated with activation of Akt1 but not Akt2 and Akt3 kinases in contracting muscles.