DNA samples from five unrelated Japanese patients with 21-hydroxylase (21-OHase) deficiency were studied by Southern analysis using human 21-OHase cDNA. Patterns seen after digestion with not only TaqI but also KpnI showed that two out of the five patients were homozygous for a deletion of the 21-OHase B gene. This result supports the report that the 21-OHase B gene is functional. In the other three, smaller mutations might be responsible for the disorder. The parents of one of the two patients with the deletion had a common ancestor. Hybridization patterns of DNA from members of the family of the patient were consistent with an autosomal recessive mode of inheritance of the deletion that correlates with the clinical phenotype. The deletion segregated with HLA-Aw 24; Bw 61; Cw 3. Heterozygous carriers of 21-OHase deficiency could be detected by comparing the patterns as well as the HLA haplotypes in this family. The application of the family study to the prenatal diagnosis is also discussed.