GLUT4 translocation and activation of glucose uptake in skeletal muscle can be induced by both physiological (i.e., insulin, nerve stimulation, or exercise) and pharmacological (i.e., phorbol ester) means. Recently, we demonstrated that high glucose levels may mimic the effects of phorbol esters on protein kinase C (PKC) and insulin receptor function (J Biol Chem 269:3381–3386, 1994). In this study, we tested whether the previously described effects of phorbol esters on translocation of GLUT4 in myotubes in culture and also in rat skeletal muscle might be mimicked by glucose. We found that stimulation of C2C12 myotubes with both insulin (10–7) mol/l, 5 min) and glucose (25 mmol/l, 10 min) induces a comparable increase of the GLUT4 content in the plasma membrane. To test whether this effect occurs in intact rat skeletal muscle as well, two different model systems were used. As an in vitro model, isolated rat hindlimbs were perfused for 80 min with medium containing 6 mmol/l glucose ± insulin (1.6 × 10–9 mmol/l, 40 min) or 25 mmol/l glucose. As an in vivo model, acute hyperglycemia (> 11 mmol/l glucose, 20 min) was induced in Wistar rats by intraperitoneal injection of glucose under simultaneous suppression of the endogenous insulin release by injection of somatostatin. In both models, subcellular fractions were prepared from hindlimb skeletal muscle, and plasma membranes were characterized by the enrichment of the marker enzyme α1 Na+ -K+ -ATPase. Acute hyperglaycemia in vivo (n = 5) and in vitro (n = 6) induced an increases of GLUT4 content in the α1 Na+ -K+ -ATPase–enriched fraction (in vivo, 2.45 ± 0.47-fold increase to basal [mean ±SE]; in vitro, 1.71 ± 0.14-fold increase to basal), which was Quantitatively similar to that obtained after insulin treatment (in vivi, 2.35 ± 0.62-fold increase to basal; in vitro, 1.91 ± 0.21-fold increase to basal). Glucose-in-duced GLUT4 translocation in myotubes was prevented by prior addition of the PKC inhibitor 1-(5-isoquinolinyl-by prior addition of the PKC inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine; in rat skeletal muscle,GLUT4 translocation was paralleled by a translocation of PKC β, while no effect on PKC α, δ, ∊, and ζ was observed. These results suggest that glucose-induced GLUT4 translocation might represenet an insulin-independent autoregulatory mechanism of the skeletal muscle to rapidly increase glucose uptake in acute hyperglycemia. Activation of PKC β might be involved in this mechanisum in skeletal muscle. GLUT4 translocation was paralleled by a translocation of PKC β, while no effect on PKC α, δ, μ, and ¶ was observed. These results suggest that glucose-induced GLUT4 translocation might represent an insulin-independent autoregulatory mechanism of the skeletal muscle to rapidly increase glucose uptake in acute hyperglycemia. Activation of PKC P might be involved in this mechanism in skeletal muscle.