A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, co-expressing glycerol dehydrogenase (GlyDH) from Gluconobacter oxydans and glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, has been successfully constructed and used for the reduction of aliphatic aldehydes, such as hexanal or glyceraldehyde to the corresponding alcohols. This catalyst was characterized in terms of growth conditions, temperature and pH dependency, and regarding the influence of external cofactor and permeabilization. In the case of external cofactor addition we found a 4.6-fold increase in reaction rate caused by the addition of 1 mM NADP+. Due to the fact that pH and temperature are also factors which may affect the reaction rate, their effect on the whole-cell catalyst was studied as well. Comparative studies between the whole-cell catalyst and the cell-free system were investigated. Furthermore, the successful application of the whole-cell catalyst in repetitive batch conversions could be demonstrated in the present study. Since the GlyDH was recently characterized and successfully applied in the kinetic resolution of racemic glyceraldehyde, we were now able to transfer and establish the process to a whole-cell system, which facilitated the access to L-glyceraldehyde in high enantioselectivity at 54% conversion. All in all, the whole-cell catalyst shows several advantages over the cell-free system like a higher thermal, a similar operational stability and the ability to recycle the catalyst without any loss-of-activity. The results obtained making the described whole-cell catalyst an improved catalyst for a more efficient production of enantiopure L-glyceraldehyde. Biotechnol. Bioeng. 2010;106: 541–552. © 2010 Wiley Periodicals, Inc.