The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number redox-inactive ketoreductase (KR) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR degrees domains is, as predicted, a (2R)-2-methyl-3ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoylACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-H-2]-(2R,3S)-2-methyl-3hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP(+) in the presence of redox-inactive, recombinant NanKR1 degrees or NanKRS degrees, from modules 1 and 5 of the nanchangmycin synthase, or recombinant Sa1KR7 degrees from module 7 of the salinomycin synthase, resulted in first order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR degrees catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2-H-2]-(2R)-2-methyl-3ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR degrees domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR degrees domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.