We set up a rapid and highly sensitive sandwich enzyme immumoassay (EIA) of human insulin using anti-porcine insulin guinea pig Fab'-peroxidase conjugate, and polystyrene balls or polystyrene plates coated with anti-porcine insulin 1gG. The minimal detectable quantity was 0.15μU/ml and O.63μU/ml using 20μ1 sample in polystyrene ball method and polystyTene plate methed, respectively All procedures were completed within three hours. Gel chromatographic analysis of recombinant human insulin showed a single peak of imunoreactive insulin, whereas two peaks, corresponding to insulin and proinsulin, were obtained from plasma obtained from a patient with myotonic dystrophy. The diluted human plasma sample showed linear response parallel lo the standard. Plasma immumoreactive insulin levels measured by EIA were well correlated with those of radioimmunoassay. The EIA systems are accurate enough to measure plasma immunoreactive insulin levels within a few hours without any concentration, suggesting a useful tool for evaluating insulin secretion in clinical and basic research.