To determine the effects of prolonged elevation of plasma free fatty acids (FFAs) on insulin secretion, we infused Liposyn II (4.3 μmol · kg⁻¹ · min⁻¹) plus heparin (0.4 U · kg⁻¹ · min⁻¹) intravenously into six healthy volunteers for 48 h. Another six volunteers received saline infusions and served as control subjects. In all 12 subjects (11 men and 1 woman), plasma glucose was clamped at ∼8.6 mmol/l. Liposyn/heparin infusion resulted in a 9.4-fold increase in plasma FFA concentration (from 132 to 1,237 μmol/l), a 46% increase in insulin secretion rates (from 241 to 352 pmol/min, P < 0.05) (determined by deconvolution of plasma C-peptide concentration), and a 30% decrease, during the initial 24 h, in the rate of glucose infusion needed to maintain hyperglycemia (from 55.5 to 39.1 μmol · kg⁻¹ · min⁻¹, P < 0.02). This decrease disappeared during the second 24 h. In summary, we found that physiologically elevated plasma FFAs 1) potentiated glucose-stimulated insulin secretion for 48 h and 2) initially caused peripheral insulin resistance that disappeared during the 2nd day, probably as a result of elevated circulating insulin levels. We conclude that in healthy volunteers under hyperglycemic conditions, fat infusion produced insulin resistance that was compensated for after ∼24 h by persistent hypersecretion of insulin.