Six healthy human subjects were studied during three 75-g oral, [¹³C]glucose tolerance tests to assess the kinetics of dexamethasone-induced impairment of glucose tolerance. On one occasion, they received dexamethasone (4 × 0.5 mg/day) during the previous 2 days. On another occasion, they received a single dose (0.5 mg) of dexamethasone 150 min before ingestion of the glucose load. On the third occasion, they received a placebo. Postload plasma glucose was significantly increased after both 2 days dexamethasone and single dose dexamethasone compared with control (P < 0.05). This corresponded to a 20-23% decrease in the metabolic clearance rate of glucose, whereas total glucose turnover ([6,6-²H]glucose), total (indirect calorimetry) and exogenous glucose oxidation (¹³CO₂ production), and suppression of endogenous glucose production were unaffected by dexamethasone. Plasma insulin concentrations were increased after 2 days of dexamethasone but not after a single dose of dexamethasone. In a second set of experiments, the effect of a single dose of dexamethasone on insulin sensitivity was assessed in six healthy humans during a 2-h euglycemic hyperinsulinemic clamp. Dexamethasone did not significantly alter insulin sensitivity. It is concluded that acute administration of dexamethasone impairs oral glucose tolerance without significantly decreasing insulin sensitivity.