Highly specific immunoradiometric assays were used to measure plasma concentrations of insulin, proinsulin, and 32-33 split proinsulin in neonates (n = 16). Neonatal plasma insulin concentrations were high relative to blood glucose concentrations and compared with adult insulin-glucose relations. Concentrations of proinsulin and 32-33 split proinsulin together accounted for 34-70% of the total concentration of insulin and pro-peptides. This study confirms the need to use a specific assay and neonatal reference data in the diagnosis of neonatal hyperinsulinism, and shows that neonatal pancreatic beta cell function may differ from that of older subjects.