The relative role of contractions, insulin, and increased supply of glucose and insulin, via an increase in blood flow, in stimulating glucose uptake in skeletal muscle during contractions was studied in isolated perfused rat hindlimbs. ffindlimbs were perfused with a standard per-fusate medium containing 6 mmol/1 glucose and four different insulin concentrations (0, 100, 500, and 20,000 μU/ml). Contractions were induced by supramaximal intermittent electrical stimulation of the sciatic nerve. Three different perfusion protocols were used: 1) muscles were stimulated to contract without concomitantly increasing perfusate flow; 2) flow was increased in the absence of electrical stimulation; and 3) muscles were stimulated to contract together with a flow increase. Both contractions and increased flow of perfusate, applied as separate stimuli, increased (P < 0.05) glucose uptake in the absence of insulin. Yet when submaximal insulin concentrations were added to the perfusate, the stimulatory action of both contractions and increased blood flow on muscle glucose uptake was augmented. The higher the submaximal insulin concentration, the greater the increment (P < 0.05). This effect, however, faded at supra-maximal insulin concentration. Electrical stimulation associated with an increase in perfusion flow rate produced a greater (P < 0.05) rise in glucose uptake than did contractions alone. In fact, stimulation of muscle glucose uptake by contractions and increased flow proved to be additive at any insulin concentration. We conclude that contractions and increased blood flow act as additional stimuli to muscle glucose uptake at any insulin concentration. Furthermore, insulin appears to be a major contributor to stimulating glucose uptake rate in muscle during contractions by facilitating both the flow-induced and the contraction-induced increment in muscle glucose uptake.