Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during hyperinsulinemia, because glucose phosphorylation is the more formidable barrier to insulin-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair insulin-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity is increased. Thus, chow-fed C57BL/6J mice with a GLUT4 partial knockout (GLUT4(+/-)), hexokinase II overexpression (HK(Tg)), or both (HK(Tg) + GLUT4(+/-)) and wild-type littermates were studied. Carotid artery and jugular vein catheters were implanted for sampling and infusions at 4 months of age. After a 5-d recovery, 5-h fasted mice (n = 8-11/group) underwent a 120-min saline infusion or insulin clamp (4 mU/kg.min insulin with glucose maintained at 165 mg/dl) and received a 2-deoxy[(3)H]glucose bolus to provide an index of MGU (R(g)) for the soleus, gastrocnemius, and superficial vastus lateralis. Basal R(g) from all muscles studied from saline-infused mice were not changed by any of the genetic modifications. HK(Tg) mice had augmented insulin-stimulated R(g) in all muscles studied compared with remaining genotypes. Insulin-stimulated R(g) was not impaired in any of the muscles studied from GLUT4(+/-) mice. However, the enhanced insulin-stimulated R(g) created by HK overexpression was ablated in HK(Tg) + GLUT4(+/-) mice. Thus, a 50% reduction of normal GLUT4 content in the presence of normal HK activity does not impair insulin-stimulated MGU. However, when the glucose phosphorylation barrier is lowered by HK overexpression, GLUT4 availability becomes a limitation to insulin-stimulated MGU.