Amniotic fluid binding protein (AFBP) is a heat and acid stable somatomedin (Sm)-binding protein with a mol wt of 35-40,000 and an isoelectric point of +/- 4.7. It is reactive in RRAs for Sm and inhibits Sm activity in Sm bioassays. AFBP was purified from midgestational human amniotic fluid (AF) using acid-ethanol extraction, Sephadex G-150 chromatography, high speed gel filtration chromatography, and disc gel-electrophoresis. Specific binding activity (microgram equivalents per mg protein) was quantitated by incubation with 125I-insulin-like growth factor II and dextran-coated charcoal separation. Protein recovery was less than 1%. AFBP antiserum was produced by immunizing rabbits with purified AFBP. The antiserum was cleared of human serum albumin antibodies by affinity chromatography. Immunoelectrophoresis of 20x concentrated preterm AF and fetal serum resulted in one precipitin line. AFBP was labeled by the chloramine-T method. The AFBP antiserum specifically bound +/- 35% of added 125I-AFBP at a final dilution of 1:5000. A double antibody RIA was developed. The AFBP level measured by RIA in midgestation AF (n = 30) was 148 +/- 18 (SEM) and in term AF (n = 12) 72 +/- 36 mu geq/ml. Insulin-like growth factor I/Sm-C values (determined by RIA) in the same samples were uniformly very low (less than 0.10 U/ml). When serum was chromatographed on Sephadex G-200 at pH 2.2, AFBP-RIA activity eluted in one peak corresponding to a mol wt of 35-40,000. Highest activity was found in fetal serum (gestational age +/- 20 weeks) and lowest in serum from adults. The development of the AFBP-RIA may contribute to further elucidation of the physiological importance of Sm and the Sm-binding proteins in pre- and postnatal growth.