11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) catalyzes the conversion of physiological glucocorticoids to inactive products, thus protecting nonselective renal mineralocorticoid receptors from circulating glucocorticoids (ensuring aldosterone selectivity in vivo) and modulating glucocorticoid access to mineralocorticoid receptors and glucocorticoid receptors in other tissues. Detection of multiple mRNA and immunoreactive 11 beta-OHSD species in kidney, but not liver, extracts suggests the presence of tissue-specific isoforms. To determine whether differential promoter usage might explain the mRNA heterogeneity we cloned and sequenced rat 11 beta-OHSD genomic DNA. Total identity was found between the nucleotide sequence of exons 1 and 2 and the previously published rat liver cDNA. Using both primer extension and RNase protection analyses we found the predominant transcription start site in liver (+1) is 105 base pairs (bp)5' of the start of translation. In kidney two additional Cap sites were detected: 1) 264 bp 5' of exon 1; there is no in-phase open reading frame, suggesting the additional 5' sequence is not translated; and 2) 65 bp upstream of exon 2, within intron A; the predicted truncated protein lacks the first 26 hydrophobic residues. Oligonucleotide probes specific to transcripts arising from each promoter confirmed that all three are employed in kidney, whereas a single predominant species was found in liver, thus demonstrating tissue-specific differential promoter usage of the 11 beta-OHSD gene.