While the cis-acyltransferase modular polyketde synthese assembly lines have largely structurally dissected. enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observe crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacilacene nonribosomal peptide syrithetaseipolyketide synthase at 2.35 angstrom resolution. This KR naturally reduces both alpha- and beta-keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N-acetylcysteamine-bound beta-keto substrate to a alpha-beta-hydroxy product, but also an N-acetylcysteamine-botind or-keto substrate to an alpha,beta-hydrorq, product. That the substrates must enter the active site from opposite directions to generate these stereo chemistries suggests that the acyl-phosphrlpalitetheine moiety is capable of accessing very different conformations despite being anchored to a serilie residue of a docked acyl carrier protein. The features enabling stereocontrolled alpha-ketoreduction may not be extensive since a KR that naturally reduces a p-keto group within a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same a-keto substrate to generate the nu,alpha-hdrorry product. A sequence analysis of trans-acyltransferase KRs reveals that a single residue, rather than a three-residue motif found in cis-acyltransfemse KRs, is predictive of the orientation of the resulting beta-hydroxyl group.