Twenty-one hydroxylase (P450c21) is a key enzyme essential for normal zona glomerulosa and fasciculata function. Recently, 21-hydroxylase deficiency has been implicated in the pathogenesis of adrenocortical tumors. Therefore, we investigated the mutational spectrum of the CYP21B gene and the messenger RNA expression of P450c21 in six aldosterone-producing adenomas, seven cortisol-producing adenomas, two nonfunctional incidentally detected adenomas, and four adrenal carcinomas. DNA from leukocytes and tumors was amplified by PCR using primers specific for the CYP21B gene. The 10 exons, intron 2, intron 7, all other exon/intron junctions, and 380 bp of the promoter region of CYP21B were automatically sequenced. Poly(A) RNA was extracted from tumor tissue, dot blotted on a nylon membrane, and hybridized with 32P-labeled P450 side-chain cleavage, P450 17-alpha-hydroxylase, and P450c21 complementary DNA probes. We detected heterozygous germline mutations (exon 7, Val 281Leu) in two patients, one with a cortisol-producing adenoma and the other with an androgen-secreting adrenocortical carcinoma. A somatic, heterozygous microdeletion was found in exon 3 of one aldosterone-producing adenoma. The P450c21 gene expression correlated with the clinical phenotype of the tumor, with low P450c21 messenger RNA expression in nonfunctional adenomas (18.8%, 1.5%) compared with high P450c21 expression in aldosterone- and cortisol-producing adenomas (84 +/- 8% and 101 +/- 4%, respectively, vs. normal adrenals, 100 +/- 10%). In conclusion, the prevalence of heterozygous germline mutations in the CYP21B gene was higher in patients with adrenocortical tumors (11%; 95% confidence interval, 1-34%) than in the general European population (2%; 95% confidence interval, 1.93-2.06%), but this difference is questionable because of the low number of subjects in our series. The pathophysiological significance of this finding in the presence of one normal CYP21B gene seems to be low, suggesting that 21-hydroxylase deficiency is not a major predisposing factor for adrenal tumor formation.