A CYP51-like gene (Z80226) of Mycobacterium tuberculosis was expressed in Escherichia coli. The product exhibited absorption spectra characteristic of P450. The expressed P450 formed a stoichiometric complex with ketoconazole, one of the specific ligands of CYP51. These findings indicate that the CYP51-like gene of M. tuberculosis actually encodes a P450 having active-site environments similar to those of CYP51, confirming the predicted orthologous nature of this gene to eukaryotic CYP51. Although eukaryotic CYP51s are membrane-binding proteins, the expressed product was accumulated only in the soluble fraction of the host cells.