An enzyme of Ralstonia/Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity, It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH). Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa. It contained molybdenum. iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme. The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide. Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme. Purified NDH exhibited a maximal specific activity of 22.6 mumol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein. using nicotinic acid as electron donor. The apparent K-m value for nicotinic acid was determined to be 154 muM. Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima. Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O-2 NDH was inactivated upon incubation with cyanide. but no loss of activity was obtained in the presence of arsenite.