The effects of insulin and contraction on glucose transport and metabolism were investigated in rat epitrochlearis muscles in vitro. Insulin dose-response curves showed a threshold (approximately 50 microunits/ml) and saturation-type (approximately 1 mU/ml) kinetics, whereas isometric contraction activated glucose transport and metabolism in a linear fashion with no evidence of a threshold. Insulin and contraction increased the apparent maximal rate of uptake of the hexose transport system with minimal effect on its apparent Km. The stimulatory effects of insulin and contraction were additive; similar results were obtained with 2-deoxy-D-glucose. Contraction stimulated glucose transport in three different preparations of muscles depleted of insulin: 1) exhaustively washed for 2 h, 2) rats infused with anti-insulin serum, and 3) chronically (streptozotocin-induced) diabetic rats. Prostaglandin E2 augmented the effect of a submaximal concentration of insulin on glucose transport without exerting any effect by itself but had no effect on contraction-augmented glucose transport. It is concluded that insulin and contraction activate glucose transport and metabolism via independent mechanisms.