One challenge to the development of new antitubercular drugs is the existence of multiple virulent strains that differ genetically. We and others have recently demonstrated that CYP125A1 is a steroid C(26)-monooxygenase that plays a key role in cholesterol catabolism in Mycobacterium tuberculosis CDC1551 but, unexpectedly, not in the M. tuberculosis H37Rv strain. This discrepancy suggests that the H37Rv strain possesses compensatory activities. Here, we examined the roles in cholesterol metabolism of two other cytochrome P450 enzymes, CYP124A1 and CYP142A1. In vitro analysis, including comparisons of the binding affinities and catalytic efficiencies, demonstrated that CYP142A1, but not CYP124A1, can support the growth of H37Rv cells on cholesterol in the absence of cyp125A1. All three enzymes can oxidize the sterol side chain to the carboxylic acid state by sequential oxidation to the alcohol, aldehyde, and acid. Interestingly, CYP125A1 generates oxidized sterols of the (25S)-26-hydroxy configuration, whereas the opposite 25R stereochemistry is obtained with CYP124A1 and CYP142A1. Western blot analysis indicated that CYP124A1 was not detectably expressed in either the H37Rv or CDC1551 strains, whereas CYP142A1 was found in H37Rv but not CDC1551. Genetic complementation of CDC1551 Δcyp125A1 cells with the cyp124A1 or cyp142A1 genes revealed that the latter can fully rescue the growth defect on cholesterol, whereas cells overexpressing CYP124A1 grow poorly and accumulate cholest-4-en-3-one. Our data clearly establish a functional redundancy in the essential C(26)-monooxygenase activity of M. tuberculosis and validate CYP125A1 and CYP142A1 as possible drug targets.