Two NAD-dependent dehydrogenases which oxidize secondary alcoholic groups at the Cα-position of dimeric lignin model compounds were purified from Pseudomonas sp. TMY1009. These enzymes have been designated Cα-dehydrogenase I and II (DH-I and DH-II). DH-II was purified to electrophoretic homogeneity. The molecular weight of DH-II, which is composed of four identical subunits, is 125, 000. DH-I was partially purified and the molecular weight of DH-I is 94, 000. Both DH-I and DH-II are active for three kinds of dimeric lignin model compounds related to major lignin substructures, although their specificities and affinities are different.