Samples of human amniotic fluid were obtained by amniocentesis prior to delivery and stored at −15 °C until analysed for 17β-hydroxyandrogens. Conjugated androgens were hydrolysed by boiling with concentrated hydrochloric acid and the free steroids extracted with diethyl ether. The dried extracts were then subjected to radioimmunoassay by the technique of Chen, Zorn, Hallberg & Wieland (1971), except that dextran-coated charcoal was used to separate the free testosterone. Since the antiserum (kindly donated by Dr M. C. Hallberg, Cleveland) was prepared against testosterone-3-bovine serum albumin and it cross-reacted significantly only with 17β-hydroxy-5α-androstan-3-one, the method must be considered as measuring total immunoreactive 17β-hydroxyandrogens though testosterone is probably the major one present. Comparison of this method with a protein-binding method for testosterone involving a prior chromatographic step, gave a correlation coefficient, r, of 0·973. Other steroids are therefore not likely to contribute to the estimation of 17β-hydroxyandrogens. Water and solvent blanks were