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In this study, we developed a measuring system for proinsulin in human serum. Insulin-like substances (ILS) were isolated from serum by an affinity chromatography, and proinsulin in ILS was separated from insulin by high-performance liquid chromatography (HPLC). For the radioimmunoassay (RIA), porcine proinsulin was used as the standard and as 125I-labelled antigen, and porcine insulin anti-serum was as the antibody. Proinsulin-like immunoreactivity (PLI) can be estimated directly from a standard curve, and the detection limit of PLI is 0.0044 pmol/ml. By this method, the values of PLI and insulin-like immunoreactivity (IRI) in human blood were able to estimate precisely. PLI values in the fasting state and in 30, 60, 90, and 120 min after oral glucose stimulation from nine healthy subjects were 0.011 +/- 0.002, 0.045 +/- 0.014, 0.078 +/- 0.019, 0.064 +/- 0.016, and 0.027 +/- 0.006 pmol/ml, respectively. The increase in PLI response was observed later than that in IRI response. PLI/total IRI ratio in the fasting state and 60 min after oral glucose were 24 and 36% respectively.