An isotopic method was used in conscious rats to determine the roles of glucose transport and the transsarcolemmal glucose gradient (TSGG) in control of basal and insulin-stimulated muscle glucose uptake. Rats received an intravenous 3-O-[3H]methylglucose (3-O-[3H]MG) infusion from -100 to 40 min and a 2-deoxy-[3H]glucose infusion from 0 to 40 min to calculate a glucose metabolic index (Rg). Insulin was infused from -100 to 40 min at rates of 0.0, 0.6, 1.0, and 4.0 mU.kg-1.min-1, and glucose was clamped at basal concentrations. The ratios of soleus intracellular to extracellular 3-O-[3H]MG concentration and soleus glucose concentrations were used to estimate the TSGG using principles of glucose counter-transport. Tissue glucose concentrations were compared in well-perfused, slow-twitch muscle (soleus) and poorly perfused, fast-twitch muscle (vastus lateralis, gastrocnemius). Data show that 1) small increases in insulin increase soleus Rg without decreasing TSGG, suggesting that muscle glucose delivery and phosphorylation can accommodate the increased flux; 2) due to a limitation in soleus glucose phosphorylation and possibly delivery, insulin at high physiological levels decreases TSGG, and at supraphysiological insulin levels the TSGG is not significantly different from 0; 3) maximum Rg is maintained even though TSGG decreases with increasing insulin levels, indicating that glucose transport continues to increase and is not rate limiting for maximal insulin-stimulated glucose uptake; and 4) muscle consisting of fast-twitch fibers that are poorly perfused exhibits a 35-45% fall in tissue glucose with insulin, suggesting that glucose delivery is a major limitation in sustaining the TSGG. In conclusion, control of glucose uptake is distributed between glucose transport and factors that determine the TSGG. Insulin stimulation of glucose transport increases the demands on the factors that maintain glucose delivery to the muscle membrane and glucose phosphorylation inside the muscle.