The aim of this study was to determine barriers limiting muscle glucose uptake (MGU) during increased glucose flux created by raising blood glucose in the presence of fixed insulin. The determinants of the maximal velocity (V(max)) of MGU in muscles of different fiber types were defined. Conscious rats were studied during a 4 mU x kg(-1) x min(-1) insulin clamp with plasma glucose at 2.5, 5.5, and 8.5 mM. [U-(14)C]mannitol and 3-O-methyl-[(3)H]glucose ([(3)H]MG) were infused to steady-state levels (t = -180 to 0 min). These isotope infusions were continued from 0 to 40 min with the addition of a 2-deoxy-[(3)H]glucose ([(3)H]DG) infusion. Muscles were excised at t = 40 min. Glucose metabolic index (R(g)) was calculated from muscle-phosphorylated [(3)H]DG. [U-(14)C]mannitol was used to determine extracellular (EC) H(2)O. Glucose at the outer ([G](om)) and inner ([G](im)) sarcolemmal surfaces was determined by the ratio of [(3)H]MG in intracellular to EC H(2)O and muscle glucose. R(g) was comparable at the two higher glucose concentrations, suggesting that rates of uptake near V(max) were reached. In summary, by defining the relationship of arterial glucose to [G](om) and [G](im) in the presence of fixed hyperinsulinemia, it is concluded that 1) V(max) for MGU is limited by extracellular and intracellular barriers in type I fibers, as the sarcolemma is freely permeable to glucose; 2) V(max) is limited in muscles with predominantly type IIb fibers by extracellular resistance and transport resistance; and 3) limits to R(g) are determined by resistance at multiple steps and are better defined by distributed control rather than by a single rate-limiting step.