The steroid binding CYP142 cytochrome P450 enzymes of Mycobacterium species are involved in the metabolism of cholesterol and its derivatives. The equivalent enzyme from Mycobacterium ulcerans was studied to compare the degree of functional conservation between members of this CYP family. We compared substrate binding of the CYP142A3 enzymes of M. ulcerans and M. marinum and CYP142A1 from M. tuberculosis using UV-vis spectroscopy. The catalytic oxidation of cholesterol derivatives by all three enzymes was undertaken. Both CYP142A3 enzymes were structurally characterized by X-ray crystallography. The amino acid sequences of the CYP142A3 enzymes are more similar to CYP142A1 from M. tuberculosis than CYP142A2 from Mycolicibacterium smegmatis. Both CYP142A3 enzymes have substrate binding properties, which are more resemblant to CYP142A1 than CYP142A2. The cholest-4-en-3-one-bound X-ray crystal structure of both CYP142A3 enzymes were determined at a resolution of