Insulin, like other secretory peptides, is synthesized via a larger and less active precursor, proinsulin, converted in the beta cell by sequential limited proteolysis to insulin and C-peptide which are stored in secretory granules. Since this process is incomplete, some intact and partially processed proinsulins with variable biological and immunological activities remain trapped in the granules and enter the circulation with insulin, resulting in the heterogeneity of plasma immunoreactive insulin (IRI). Whereas methods measuring proinsulin from corrected IRI in sera fractionated by gel chromatography were not sufficiently sensitive and specific, immunoradiometric assays (IRMA) now allow reliable determinations of proinsulin, split proinsulins and true insulin and thereby the monitoring of the dynamics of conversion in various diabetic states. The recent finding of increased 32,33-split proinsulin associated with absolute true insulin deficiency, correlated with cardiovascular risk factors in Type II diabetics, sheds new light on the molecular pathology of noninsulin-dependent diabetes.