Exercise increases permeability of muscle to glucose. Normally, the effects of exercise and a maximal insulin stimulus on glucose transport are additive. However, the combined effect on rat epitrochlearis muscle permeability to 3-O-methylglucose (3-MG) of a maximal insulin stimulus followed by in vitro contractile activity of 1.24 +/- 0.06 mumol.10 min-1.ml intracellular water-1 was no greater than that of either stimulus alone. We found that this absence of an additive effect was caused by prolonged exposure to an unphysiologically high insulin concentration (20,000 microU/ml for 60 min), which, in addition to stimulating glucose transport, appears to prevent further increases in permeability to glucose. When the treatments were reversed and muscles were first stimulated to contract and then incubated with 20,000 microU/ml insulin, 3-MG uptake (mumol.10 min-1.ml intracellular water-1) increased from a control value of 0.26 +/- 0.03 to 1.80 +/- 0.15, compared with 1.04 +/- 0.06 for contractile activity alone, 1.21 +/- 0.08 for insulin, and 1.88 +/- 0.11 for exercise (swimming) plus insulin. Swimming plus in vitro contractile activity did not have a greater effect than contractile activity alone. Our results provide evidence that 1) the effect of exercise on muscle permeability to glucose is mediated solely by a process associated with contractile activity, and 2) it is advisable to avoid the use of unphysiologically high insulin concentrations in studies designed to elucidate in vivo actions of insulin.