Substrate specificity in the aldo-keto reductase (AKR) superfamily is determined by three mobile loops positioned at the top of the canonical (α/β)8-barrel structure. These loops have previously been demonstrated to be modular in a well-studied class of AKRs, in that exchanging loops between two similar hydroxysteroid dehydrogenases resulted in a complete alteration of substrate specificity (Ma,H. and Penning,T.M. (1999) Proc. Natl Acad. Sci. USA, 96, 11161–11166). Here, we further examine the modularity of these loops by grafting those from human aldose reductase (hAR) into the hyperthermostable AKR, alcohol dehydrogenase D (AdhD), from Pyrococcus furiosus. Replacement of Loops A and B was sufficient to impart hAR activity into AdhD, and the resulting chimera retained the thermostability of the parent enzyme. However, no active chimeras were observed when the hAR loops were grafted into a previously engineered cofactor specificity mutant of AdhD, which displayed similar kinetics to hAR with the model substrate DL-glyceraldehyde. The non-additivity of these mutations suggests that efficient turnover is more dependent on the relative positioning of the cofactor and substrate in the active site than on binding of the individual species. The ability to impart the substrate specificities of mesostable AKRs into a thermostable scaffold will be useful in a variety of applications including immobilized enzyme systems for bioelectrocatalysis and fine chemical synthesis.