The effect of ascorbic acid on glucose-induced insulin release from single pancreatic islets was measured using a new, ultra-sensitive enzyme-linked immunosorbent insulin assay. Within 20 s ascorbic acid inhibited insulin secretion; inhibition was dose dependent and completely reversible. There was a 50% inhibition of the secretory response with 200 microM ascorbic acid and 90% inhibition with 400 microM ascorbic acid. The decrease in insulin secretion was recorded as a reduction of the amplitudes of the fast insulin transients, which give rise to the oscillatory nature of insulin secretion. The inhibition of glucose-induced insulin release by ascorbic acid was associated with hyperpolarization of the pancreatic beta-cell. Suppression of glucose-induced membrane depolarization was evident after 20 s, was dose dependent, and was completely reversible. The data here may provide the first explanation of why plasma ascorbate concentrations are tightly controlled.