The effect of glucocorticoids on adipose tissue lipolysis in animals and humans is controversial. To determine whether a physiological increase in plasma cortisol, similar to that observed in diabetic ketoacidosis and other stress conditions, stimulates lipolysis, palmitate kinetics were measured in seven nondiabetic volunteers on two occasions with [1-¹⁴C]palmitate as a tracer. Subjects received a 6-h infusion of either 2 μg · kg⁻¹ · min⁻¹ hydrocortisone or saline in random order. On both occasions, a pancreatic clamp (0.12 μg · kg⁻¹ · min⁻¹ somatostatin, 0.05 mU · kg⁻¹ · min⁻¹ insulin, and 3 ng · kg⁻¹ · min⁻¹ growth hormone) was used to maintain plasma hormone concentrations at desired levels. Plasma cortisol concentrations increased to ∼970 nM during cortisol infusion. Palmitate rate of appearance (R_a) and concentration increased by ∼60% during cortisol infusion but did not change during saline infusion. Increments in palmitate R_a and concentration over the 6-h study were significantly greater during cortisol than saline infusion when compared by area-under-the-curve analysis (152 ± 52 vs. −48 ± 23 μmol · kg⁻¹ and 12.2 ± 4.1 vs. −4.9 ± 4.1 mmol · min⁻¹ · L⁻¹, respectively; P < 0.02). Plasma glucose concentrations did not change significantly during cortisol (5.0 ± 0.3 vs. 6.1 ± 0.6 mM, NS) or saline (4.9 ± 0.2 vs. 4.9 ± 0.1 mM, NS) infusion. In nondiabetic volunteers, a 6-h cortisol infusion was associated with a 60% increase in palmitate R_a that did not occur with saline infusion. Thus, physiological hypercortisolemia may contribute to the increased rates of lipolysis observed in humans during stress.