A new radioimmunoassay for insulin-like growth factor-II (IGF-II) is described. Compared to recombinant DNA-derived IGF-II standard, the cross-reactivity of natural or recombinant IGF-I was less than 1%. The ED50 for IGF-II standard was 1.0 ng/ml, and the mean IGF-II level in acid-ethanol-extracted serum from healthy adults was 525 +/- 87 ng/ml (SD, n = 30). Addition of the IGF binding protein IGFBP-1 (BP-28, PP12) caused dose-dependent inhibition of IGF-II tracer binding to antiserum, increasing to greater than 90% inhibition at 400 ng/ml IGFBP-1. In contrast, the IGF binding protein IGFBP-3 (BP-53) caused approximately 30% inhibition of tracer binding at 20 ng/ml IGFBP-3, with no further inhibition up to 400 ng/ml IGFBP-3. The influence of added IGF binding proteins on IGF-II displacement curves varied depending on both the type and concentration of binding protein added. It is concluded that interference in IGF radioimmunoassays by IGF binding proteins depends both on the types of binding proteins present, and on the IGF concentration, in the test samples.