Background: The presence and pathophysiological role of CYP11B1 (11β-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. Methods: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17α-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tissues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. Results: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P < 0.05) in APA. The levels of CYP11B2 mRNA were lower (P < 0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. Conclusions: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tissues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11β-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.