Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (ELX) assay has now established that such "KR0" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-H-2]-(2R,35)-2-methyl-3-hydroxypentanoyl-SACP ([2-H-2]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0) -catalyzed epimerization of transiently generated [2-H-2]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1 mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.