The sensitive and specific immunoradiometric assay is described for human proinsulin and its intermediate peptides (65-66 split and 32-33 split proinsulin). We developed a monoclonal antibody-based two-site immunoradiometric assay with use of streptavidin-biotin labeling. The detection limits of the assays lie in the range of 0.5-2.0 pM. In the proinsulin assay proinsulin cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. In the assay of 65-66 split proinsulin it does not cross-react with insulin, proinsulin or 32-33 split proinsulin. In the 32-33 split proinsulin assay it cross-reacted 84% with proinsulin and 60% with 65-66 split proinsulin. The precision (C.V.) of the assays was less than 15% over the various concentration. The mean concentrations of insulin, proinsulin, 65-66 split proinsulin and 32-33 proinsulin in eight young male subjects in the fasting state were (pM ± S.E.M.) 20 ± 3.6, 2.3 ± 0.3, undetectable (less than 1.0) and 2.1 ± 0.7 and at the maximum reached during an oral glucose tolerance test, 150 ± 26, 9.9 ± 1.4, 3.8 ± 0.6 and 19.7 ± 6.0 respectively.