Amniotic fluid levels of 21-deoxycortisol (21-DOF) and 17-hydroxyprogesterone (17-OHP) were measured in 49 pregnancies, including 31 pregnancies at risk for CAH. The results were compared with those obtained by HLA typing and linkage analysis to a HLA DNA probe. The mean amniotic fluid levels in the control pregnancies were 0.28 nmol/L for 21-DOF and 4.1 nmol/L for 17-OHP. The levels were similar in early and midpregnancy for 21-DOF (0.29 vs. 0.27 nmol/L) and 17-OHP (3.4 vs. 4.2 nmol/L). The amniotic fluid 21-DOF level was 1.75 nmol/L in affected pregnancies, significantly higher than in the control pregnancies (mean, 0.28 nmol/L). The mean amniotic fluid 17-OHP level in the affected pregnancies (30.5 nmol/L) also was significantly higher than that in the control pregnancies (4.10 nmol/L). Simultaneous measurement of 21-DOF and 17-OHP levels in amniotic fluid from 10-18 weeks of gestation can be used for early diagnosis of congenital adrenal hyperplasia.