Monoclonal antibody-based two-site immunoradiometric assays are described for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. The detection limits of the assays lie in the range 0.8-2.5 pM. The assays for 65-66 and 32-33 split proinsulins do not distinguish between these substances and their respective C-terminal di-desamino derivatives. The assay of 65-66 split proinsulin does not cross-react with insulin, proinsulin or 32-33 split proinsulin. This material was undetectable (less than 1.0 pM) in plasma taken after an overnight fast in eight normal male subjects and the maximum individual concentration reached in plasma taken during an oral glucose tolerance test of these subjects was 3.8 pM. The proinsulin assay cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. The 32-33 split proinsulin assay cross-reacted 84 and 60% with proinsulin and 65-66 split proinsulin respectively. The insulin assay cross-reacted 5.3, 62 and 5.0% with intact proinsulin, 65-66 split proinsulin and 32-33 split proinsulin respectively. The very low concentration of 65-66 split proinsulin meant that this derivative did not interfere significantly with the specificity of the assays of proinsulin and insulin. The concentration of 32-33 split proinsulin could be calculated by subtracting the cross-reactivity of the measured proinsulin. The mean concentrations of insulin, proinsulin and 32-33 split proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 0.3, 2.3 +/- 0.3 and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4 and 19.7 +/- 6.0 respectively.