The stereospecificity of hydrogen transfer from NADPH to the appropriate carbonyl substrate catalyzed by ketopantoic acid and ketopantoyl acid and ketopantoyl lactone reductases of yeast (Saccharomyces cerevisiae) and Escherichia coli has been determined. Yeast and E. coli ketopantoic acid reductases are B-specific enzymes which transfer hydrogen from [4B-3H]-NADPH to ketopantoic acid to form [3H]pantoic acid. In contrast to the usual observations on the stereospecificity of functionally similar dehydrogenases from different species, yeast and E. coli ketopantoyl lactone reductases exhibit opposite stereospecificities. Both of two forms of yeast ketopantoyl lactone reductases are A-specific enzymes which form [3H]pantoyl lactone from ketopantoyl lactone and [4A-3H]NADPH, whereas, two forms of E. coli ketopantoyl lactone reductases are B-specific enzymes.