Because of the increasing use of 17-hydroxyprogesterone (17OHP) levels with the short adrenocorticotrophic hormone (ACTH) test in the detection of 21-hydroxylase deficiency, the diagnostic efficiency of the test was evaluated in patient and family studies of congenital adrenal hyperplasia due to 21-hydroxylase deficiency and of congenital adrenal hyperplasia due to 11-hydroxylase (11OH) deficiency (the latter disorder now overlaps basally with the milder non-classical 21-hydroxylase deficiency [NC-CAH]). Stimulated 17-hydroxyprogesterone level (17OHP30), 17-hydroxyprogesterone increase (delta 17 OHP) and the ratio 17-hydroxyprogesterone increase to cortisol increase (delta 17OHP/delta cortisol) were the parameters from the short ACTH test derived for assessment. 17-OHP30 provided complete differentiation of NC-CAH from the controls and heterozygotes, but overlap between NC-CAH and 11-OH occurred. Complete differentiation of NC-CAH from 11-OH was achieved using delta 17OHP. The heterozygotes showed best differentiation from the controls using delta 17OHP/delta cortisol with a diagnostic accuracy of 70%, however marked overlap of heterozygotes and NC-CAH with 11-OH was found. The short ACTH test proved to be a valuable technique with the further detection of homozygotes (n = 3) and heterozygotes (n = 5) in the 13 families studied. However, when interpreting the short ACTH test a careful choice of parameters should be made. It should be kept in mind that mild NC-CAH patients can only be differentiated from 11OH patients by using delta 17OHP.