It was the aim of this study to determine whether prolonged hyperglycemia can produce "glucose toxicity" in normal human subjects. To this end, plasma glucose was clamped at approximately 5, approximately 8.8, and approximately 12.6 mM for 68 h in healthy volunteers. Rates of insulin secretion (by deconvolution of plasma C-peptide) and rates of insulin clearance [area under curve (AUC) 24 h insulin secretion/AUC 24 h insulin] were determined. Pre- and posthyperglycemia glucose turnover was measured (with [6,6-2H2]glucose) during euglycemic-hyperinsulinemic clamping to assess peripheral (muscle) and hepatic insulin action. Hyperglycemia (approximately 12.6 mM) for 68 h was associated with significant reductions in rates of insulin secretion (-35%, P < 0.05), insulin clearance (-57%, P < 0.05), glucose infusion rates needed to maintain hyperglycemia (-36%, P < 0.05), and insulin-stimulated glucose uptake (-55%, P < 0.01). No significant changes were seen during approximately 8.8 mM hyperglycemia or during euglycemia. These data showed that 12.6 mM hyperglycemia, but not 8.8 mM hyperglycemia or euglycemia, was associated with reduced insulin secretion, insulin clearance, and peripheral (muscle) insulin action. We concluded that 1) in normal subjects, desensitization to glucose involving beta-cells and muscle developed at plasma glucose concentrations between approximately 9 and approximately 12 mM, and 2) these effects were partially compensated for by a decrease in insulin clearance.