Insulin stimulation of glucose transport in skeletal muscle is considered to involve translocation of the skeletal muscle/adipose tissue glucose transporter isoform, Glut 4, from cytosolic vesicles to the cell surface. The current study was undertaken to investigate Glut 4 translocation in skeletal muscle of healthy volunteers during euglycaemic insulin infusion. Previous quantitative studies of glucose transport have depended on differential centrifugation methods, which demand large biopsy samples. In this study we have developed and applied a quantitative method using confocal laser microscopy, well suited to the small needle biopsies that are typically available clinically. Percutaneous biopsy of vastus lateralis skeletal muscle was performed during basal and euglycaemic insulin-stimulated conditions, and Glut 4 translocation was assessed using immunohistochemical labelling and confocal laser microscopy imaging in 14 healthy lean subjects. At physiological hyperinsulinaemia (536 +/- 16 pM), mean systemic glucose utilization was 9.27 +/- 0.78 mg/kg-min, indicative of normal insulin sensitivity. The presence of Glut 4 at the sarcolemma increased significantly (p < 0.01), with a ratio of insulin-stimulated to basal sarcolemmal Glut 4 of 1.85 +/- 0.33, indicative of insulin-stimulated Glut 4 translocation. The area of Glut 4-labelled sites also increased significantly (p < 0.01) in response to insulin infusion; this ratio was 1.56 +/- 0.13. Thus, at physiological hyperinsulinaemia, the amount of Glut 4 at the cell surface of skeletal muscle in healthy, lean individuals increases approximately twofold over basal conditions, and this process can be measured using immunohistochemical labelling imaged by confocal laser scanning microscopy.