The use of HIV protease inhibitors (PIs) has been associated with several metabolic changes, including lipodystrophy, hyperlipidemia, and insulin resistance. The etiology of these adverse effects remains unknown. PIs have recently been found to cause acute and reversible inhibition of GLUT4 activity in vitro. To determine the acute in vivo effects of indinavir on whole-body glucose homeostasis, glucose tolerance tests were performed on PI-naïve Wistar rats immediately after a single intravenous dose of indinavir. Glucose and insulin levels were significantly elevated in indinavir-treated versus control rats (P < 0.05) during the initial 30 min of the glucose tolerance test. Under euglycemic- hyperinsulinemic clamp conditions, indinavir treatment acutely reduced the glucose infusion rate required to maintain euglycemia by 18 and 49% at indinavir concentrations of 14 and 27 micromol/l, respectively. Muscle 2-deoxyglucose uptake was similarly reduced under these conditions. Restoration of insulin sensitivity was observed within 4 h after stopping the indinavir infusion. Indinavir did not alter the suppression of hepatic glucose output under hyperinsulinemic conditions. These data demonstrate that indinavir causes acute and reversible changes in whole-body glucose homeostasis in rats and support the contribution of GLUT4 inhibition to the development of insulin resistance in patients treated with PIs.