Congenital adrenal hyperplasia (CAH) is a heterogeneous group of hereditary diseases characterized by deficient adrenal cortisol synthesis. Most CAH is due to 21-hydroxylase (C21) deficiency. Genomic DNA from several families with 21-hydroxylase deficiency and normal controls was analyzed by the Southern blot hybridization technique. The restriction fragment length polymorphism (RFLP) patterns using several endonucleases, such as Taq I, Eco RI and Pvu II, at the C21 gene locus showed a very low frequency of variability in normals and most of the patients with CAH. One proband with CAH lacked the characteristic 3.7 kb Taq I fragment probed with C21 cDNA. This may be due to gene conversion and/or deletion events in the functional C21 gene locus. On the other hand, genes closely linked to the C21 locus such as C4 and HLA-DR are highly polymorphic. Using these flanking genes as probes, it is easy to perform linkage analysis and identify the inheritance trait. Prenatal diagnosis will be possible in these affected families when an additional pregnancy is expected.