We studied the contribution of the constitutive and the regulated pathways to the total secretion of islet amyloid polypeptide (IAPP) in human pancreatic islets after prolonged culture at either 5.5 or 24.4 mM glucose. In islets cultured in low concentrations of glucose, the secretion of IAPP in response to glucose was unaffected by brefeldin A (BFA) and completely blocked by ethyleneglycoltetraacetic acid. In islets cultured in high glucose concentrations, it was strongly inhibited by both agents. BFA had no effect on the glucose-induced insulin secretion. The determination of the islet peptide contents and the mRNA levels revealed a several-fold increase in the IAPP/insulin molar ratio of islets cultured in high glucose concentrations. Thus, prolonged exposure of human islets to high concentrations of glucose results in an increase in the synthesis of IAPP with respect to insulin. As a result, the release of IAPP through a mechanism sensitive to BFA is favored. These data support the hypothesis that IAPP and insulin are regulated in a noncoordinated way in human pancreatic islets.