A rapid, specific, and sensitive RIA was developed for rat corticosteroid-binding globulin (CBG). Rat CBG was purified by affinity chromatography and its precise concentration was determined by amino acid analysis. This rat CBG was injected into rabbits to raise antiserum and was used both as the assay standard and as the tracer after labeling with 125I. Antiserum to CBG was judged specific by immunoelectrophoresis and by the comparison of RIA values with steroid-binding assay values obtained following serum fractionation on ion exchange and sizing resins. The RIA was used to determine CBG levels in pregnant rats (2.65 microM on day 14 falling to 0.95 microM at parturition), their corresponding fetuses (0.24 microM on day 18 and 0.16 microM at parturition), and amniotic fluid (0.051 microM on day 13 rising to 0.21 microM on day 21).