Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD(+) and NADPH. The pnta and pntb genes encoding for the alpha- and beta-subunits were cloned and co-expressed with NADP(+)-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12 h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.