Haploid Saccharomyces cerevisiae W303-1A cells grown on different carbon sources were employed as the biocatalyst for ethyl acetoacetate reduction in n-hexane. The effects of cell immobilization on montmorillonite, as well as the addition of trehalose or sucrose solutions, were also tested. Best conversions (similar to 50%) to the chiral alcohol ethyl (S)-(+)-3-hydroxybutanoate (ee > 99%) were obtained with cells grown under respiratory metabolism with glycerol-ethanol, and higher yields were observed when trehalose was added to the reaction media. Although cells with fermentative metabolism grown on glucose were able to reduce the substrate when sucrose was added, the disaccharide was consumed by the cells during the course of the reaction, and no enantioselective product was obtained. Immobilized cells also required the addition of trehalose in order to reduce the substrate with high yield. Thus, our results indicate that trehalose may be an efficient protector of immobilized or free yeast cells during enantioselective reductions in organic solvent. (c) 2006 Elsevier Ltd. All rights reserved.