Total immunoreactive insulin (IRI) is conventionally determined by radioimmunoassays. IRI measurement in rats can be made more sensitive, accurate, and practical, as demonstrated by a new modified enzyme-linked immunosorbent assay (ELISA). It is characterized by indirect binding of an anti-insulin antibody by an antiglobulin antibody and uses the principle of competitive saturation. In this ELISA, IRI can be determined in a wide range of concentrations, corresponding to the standards. The standard curve ranges from 100 to 0.049 ng/ml IRI (1 ng/ml approximately 23.4 microU/ml approximately 172 pM rat insulin). The statistical analysis shows between- and within-assay coefficients of variation of less than or equal to 15%.