NAD(P)-dependent cholesterol dehydrogenases [NAD(P)-CDH], which allow easier quantification of cholesterol by means of directly measuring the A340 of NAD(P)H, are useful for clinical purposes. The amino acid sequences of the NH2 terminus and the fragments obtained by CNBr decomposition of the NAD(P)-CDH from a Nocardia sp. were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kbp BamHI fragment hybridizing to these probes was cloned on pUC19 in Escherichia coli. The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 364 amino acids (Mr, 39,792) and contains an NAD(P)-binding consensus sequence at its NH2-terminal portion. High-resolution S1 nuclease mapping suggested that in NAD(P)-CDH of both Nocardia and Streptomyces spp. transcription initiates at the adenine residue, which is the first position of the translational initiation triplet (AUG) of this protein. The S1 mapping experiments also showed that cholesterol-dependent regulation in the Nocardia sp. occurred at the level of transcription. In Streptomyces lividans containing the cloned fragment, however, this promoter was expressed constitutively. DNA manipulation of the cloned gene in E. coli, including the generation of a ribosome-binding sequence at an appropriate position by oligonucleotide-directed mutagenesis, led to production of this protein in a very large amount but in the enzymatically inactive form of inclusion bodies. On the other hand, a Streptomyces host-vector system was successfully used for producing 40 times as much enzymatically active NAD(P)-CDH as that produced by the original Nocardia sp.