Coenzyme B₁₂-dependent acyl-CoA mutases are radical enzymes catalyzing reversible carbon skeleton rearrangements in carboxylic acids. Here, we describe 2-hydroxyisobutyryl-CoA mutase (HCM) found in the bacterium Aquincola tertiaricarbonis as a novel member of the mutase family. HCM specifically catalyzes the interconversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA. Like isobutyryl-CoA mutase, HCM consists of a large substrate- and a small B₁₂-binding subunit, HcmA and HcmB, respectively. However, it is thus far the only acyl-CoA mutase showing substrate specificity for hydroxylated carboxylic acids. Complete loss of 2-hydroxyisobutyric acid degradation capacity in hcmA and hcmB knock-out mutants established the central role of HCM in A. tertiaricarbonis for degrading substrates bearing a tert-butyl moiety, such as the fuel oxygenate methyl tert-butyl ether (MTBE) and its metabolites. Sequence analysis revealed several HCM-like enzymes in other bacterial strains not related to MTBE degradation, indicating that HCM may also be involved in other pathways. In all strains, hcmA and hcmB are associated with genes encoding for a putative acyl-CoA synthetase and a MeaB-like chaperone. Activity and substrate specificity of wild-type enzyme and active site mutants HcmA I90V, I90F, and I90Y clearly demonstrated that HCM belongs to a new subfamily of B₁₂-dependent acyl-CoA mutases.