NAD-dependent alcohol dehydrogenase from the methanol-grown Methylcoccus sp. CRL M1 (type I membrane), Methylosinus trichosporium OB3b (type II membrane), Methylobacterium organophillum CRL 26 (type II membrane, facultative methylotroph). Pseudomonas sp. ATCC 21439, and Pichia pastoris Y-55 are secondary-alcohol-specific and that from P. pastoris Y-7556 is not. This novel secondary-alcohol-specific alcohol dehydrogenase (secondary-alcohol dehydrogenase) has been purified from methanol-grown Pseudomonas sp. ATCC 21439. Secondary-alcohol dehydrogenase shows a single protein band on acrylamide gel electrophoresis and has a molecular weight of 95000. It consists of two subunits of Mr 48000 daltons and two atoms of zinc per molecule of enzyme protein. It oxidizes secondary alcohols, notably 2-propanol and 2-butanol. Primary alcohols are not oxidized. The pH and temperature optima for secondary-alcohol dehydrogenase are 8--9, and 30--35 degrees C, respectively. The activation energy calculated is 82.8 kJ. Secondary-alcohol dehydrogenase also catalyzes the reduction of methyl ketones to their corresponding 2-alcohols in the presence of NADH (a reverse reaction). The Km values at 25 degrees C in the forward reaction for 2-butanol, (2R)-(-)-butan-2-ol, and NAD, and in the reverse reaction for 2-butanone and NADH are 2.5 x 10(-4) M, 1.6 x 10(-4) M, 11 x 10(-5) M, 1.98 x 10(-4) M, and 2.1 x 10(-6) M, respectively. The secondary-alcohol dehydrogenase activity was inhibited by metal-chelating agents and by strong thio reagents such as p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid). The substrate specificity, and mobility on gel electrophoresis of secondary-alcohol dehydrogenase and primary-alcohol dehydrogenases are compared. Secondary-alcohol dehydrogenase oxidizes preferentially the (-)-2-butanol. This is different from primary-alcohol dehydrogenase from bakers' yeast which oxidizes only the (+)-2-butanol. This may be explained in terms of the structure of the enzymes.