The phosphorylation of glucose to glucose-6-phosphate (G-6-P) is the first committed step in glucose uptake in skeletal muscle. This reaction is catalyzed by hexokinase (HK). Two HK isoforms, HKI and HKII, are expressed in human skeletal muscle, but only HKII is regulated by insulin. The present study was undertaken to determine the time course for the regulation of HK activity and expression by physiological plasma insulin concentrations in human skeletal muscle in vivo. A hyperinsulinemic-euglycemic glucose clamp and percutaneous muscle biopsy were performed in separate groups of healthy subjects after 60, 120, 180, and 360 minutes of euglycemic hyperinsulinemia. Muscle biopsies were subfractionated into soluble and particulate fractions to determine HKI and HKII activities. RNA was extracted from a separate portion of the muscle biopsy, and HKI and HKII mRNA content was determined using an RNase protection assay. Glycogen synthase (GS) activity and fractional velocity were also determined. HKII mRNA was increased 2-fold by 120 minutes and remained high versus the basal value for up to 360 minutes. HKI mRNA was unchanged throughout the study. HKII activity increased after 360 minutes of insulin infusion, and this increase was limited to the soluble fraction. In contrast, insulin induced a 1.5- to 2-fold increase in GS fractional velocity that was sustained for 360 minutes. The time course of the ability of hyperinsulinemia to increase HKII mRNA indicates that insulin is likely a physiological regulator of HKII expression in human skeletal muscle in vivo.