Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. To ascertain carrier status, adrenocorticotropin (ACTH) stimulation tests are often used. To determine the sensitivity of ACTH stimulation to detect heterozygotes and to correlate stimulated 17-hydroxyprogesterone responses with molecular genotype, we compared molecular genetic analysis of the 21-hydroxylase (CYP21) gene with 17-hydroxyprogesterone responses at 30 min in 51 individuals. Molecular genotype analysis and ACTH stimulation tests were performed in healthy volunteers (n = 20) and relatives of patients with congenital adrenal hyperplasia (n = 31). Polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, allele-specific oligonucleotide hybridization (ASOH) analysis, and restriction fragment length polymorphism (RFLP) analysis were utilized to screen for 14 CYP21 mutations which account for >90% of the mutations associated with 21-hydroxylase deficiency. Molecular genotype analysis classified 28 individuals as heterozygotic carriers and 23 individuals as normal for all mutations tested. As a group, the heterozygotes had significantly greater stimulated 17-hydroxyprogesterone responses at 10 and 30 min (P < 0.0005). However, on an individual basis, 14/28 (50%) genotyped heterozygotic carriers had stimulated 17-hydroxyprogesterone concentrations, 17-hydroxyprogesterone/cortisol ratios, and 17-hydroxyprogesterone incremental elevations indistinguishable from the genotyped normal individuals. Thus, a normal 17-hydroxyprogesterone response to ACTH stimulation testing does not exclude carrier status for 21-hydroxylase deficiency. Molecular genotype analysis is a more reliable method to determine 21-hydroxylase heterozygotes.