The chirality of the hydrogen transfer from (3R) hydroxybutyrate to NAD catalyzed by (3R)hydroxybutyrate dehydrogenase (E.C. 1.1.1.30, D-3-hydroxybutyrate : NAD oxidoreductase) from Pseudomonas lemoignei was investigated. [4(-3)H] NAD was enzymatically reduced to (4R) [4(-3)H]NADH with (3RS) hydroxybutyrate. This observation was confirmed since NAD could be reduced to (4S) [4(-3)H] NADH with (3RS) [3(-3)H] hydroxybutyrate and (3R) hydroxybutyrate dehydrogenase. From these experiments it can be concluded that (3R) hydroxybutyrate dehydrogenase from P. lemoignei should be classified as an B or (S) type dehydrogenase.