Amniotic fluid (AF) levels of all steroids leading from pregnenolone (delta 5Preg) to androgens and estrogens of both the delta 5 and the delta 4 pathways and those of cortisol and cortisone have been determined in 63 normal pregnancies (12-19 weeks gestation). The 12 unconjugated steroids [delta 5Preg, 17 alpha-hydroxypregnenolone, progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone (DHA), delta 4-androstenedione, delta 5-androstene-3 beta,17 beta-diol (delta 5Adiol), testosterone (T), estrone, 17 beta-estradiol (E2), cortisol, and cortisone] were measured by specific RIAs after appropriate purification by Celite or LH-20 column chromatography, while the sulfates of DHA and delta 5Preg were assayed directly on diluted samples. There were distinct sex differences; T and delta 4-androstenedione levels were higher (P < 0.001) in males than in females, while AF levels of delta 5Preg, 17 alpha-hydroxypregnenolone, 17-hydroxyprogesterone, DHA, and delta 5Adiol were higher (P < 0.05) in females than in males. AF levels of E2 were significantly higher in females only between 15 and 19 weeks gestation. There was no difference between sexes in AF levels of progesterone, estrone, cortisone, and cortisol. AF levels of T, delta 4-androstenedione, and E2 decreased with age in males, and AF levels of DHA increased in females during the period examined. These observed sex differences suggest that T and delta 4-androstenedione may reflect fetal testicular activity, while E2 and 17-hydroxyprogesterone might reflect fetal ovarian activity. Determination of AF levels of T would appear to be a valuable screening test for antenatal diagnosis of sex (predictive error, less than or equal to 15%), but not in the presence of steroidogenic enzyme defects. Elevated levels of 17-hydroxyprogesterone were found in the AF of two fetuses with either a 17-20 desmolase defect or 21-hydroxylase deficiency; AF levels of androgens were low in the former and high in the latter.