Using routine stable isotope dilution/gas chromatography-mass spectrometry, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, androstanediol, and 5alpha-dihydrotestosterone have been profiled in amniotic fluid of midgestation in 77 normal fetuses and 38 untreated or dexamethasone-treated fetuses at risk for 21-hydroxylase deficiency. Dexamethasone was suspended 5-7 days before amniocentesis. In normal fetuses, amniotic fluid concentrations (median, range; nanograms per mL) of 17-hydroxyprogesterone did not reveal a sex difference (1.48, 0.21-4.96), whereas those of androstenedione were lower in females (0.53, 0.00-2.71) than in males (0.93, 0.29-1.98). Testosterone levels were higher in males (0.24, 0.00-0.50) than in females (0.00, 0.00-0.27). No sex difference was found for dehydroepiandrosterone (0.47, 0.19-1.77). Levels of androstanediol and 5alpha-dihydrotestosterone were below the detection limit of our method in most cases. Regarding prenatal diagnosis of 21-hydroxylase deficiency, 17-hydroxyprogesterone and androstenedione presented the diagnostically most valuable steroids and were of equal diagnostic potential. They permitted successful diagnosis in 36 of 37 fetuses at risk: 12 were untreated and unaffected, 13 were treated and unaffected, 4 were untreated and affected (3 salt wasters and 1 simple virilizer), and 8 were treated and affected (5 salt wasters and 3 simple virilizers). In the latter group, one simple virilizer revealed normal steroid concentrations. Isotope dilution/gas chromatography-mass spectrometry, providing the highest specificity in steroid analysis, is proposed for routine use in clinical steroid analysis whenever maximal reliability is requested. Our study provides the first mass spectrometric reference data on amniotic fluid steroid concentrations and underscores the high accuracy of prenatal hormonal diagnosis of 21-hydroxylase deficiency.