The in vivo effect of elevated free fatty acids (FFA) on β-cell function in humans remains extremely controversial. We examined, in healthy young men, the acute (90 min) and chronic (48 h) effects of an approximately twofold elevation of plasma FFA vs. control on glucose-stimulated insulin secretion (GSIS). GSIS was studied in response to a graded intravenous glucose infusion (peak plasma glucose, ∼10 mmol/l,n = 8) and a two-step hyperglycemic clamp (10 and 20 mmol/l, n = 8). In the acute studies, GSIS was significantly higher, insulin sensitivity index (S_I) was lower, and disposition index (DI = insulin sensitivity × insulin secretion) was unchanged with elevated FFA vs. control [2-step clamp: DI = 8.9 ± 1.4 × 10⁻³l² ⋅ kg⁻¹ ⋅ min⁻²in control vs. 10.0 ± 1.9 × 10⁻³l² ⋅ kg⁻¹ ⋅ min⁻²with high FFA, P = nonsignificant (NS)]. In the chronic studies, there was no difference in absolute GSIS between control and high FFA studies, but there was a reduction in S_I and a loss of the expected compensatory increase in insulin secretion as assessed by the DI (2-step clamp: DI = 10.0 ± 1.2 × 10⁻³l² ⋅ kg⁻¹ ⋅ min⁻²in control vs. 6.1 ± 0.7 × 10⁻³l² ⋅ kg⁻¹ ⋅ min⁻²with high FFA, P = 0.01). In summary,1) acute and chronic FFA elevation induces insulin resistance; 2) with acute FFA elevation, this insulin resistance is precisely countered by an FFA-induced increase in insulin secretion, such that DI does not change; and 3) chronic FFA elevation disables this β-cell compensation.