Membrane-bound nicotinic acid dehydrogenase, an enzyme that catalyzes the formation of 6-hydroxynicotinic acid from nicotinic acid, was solubilized with Triton X-100, and then purified 126-fold with an 11.1% overall recovery from nicotinic acid-induced cells of Pseudomonas fluorescens TN5. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme had a molecular mass of approximately 80 kDal and consisted of one subunit. Some electron acceptors, such as phenazine methosulfate, K3Fe(CN)6 and nitro blue tetrazolium, acted as electron acceptors. The purified enzyme catalyzed the hydroxylation of nicotinic acid to 6-hydroxynicotinic acid at a rate of 672 mumol min-1 mg-1 of protein at 35-degrees-C. It also catalyzed the hydroxylation of pyrazinecarboxylic acid, 3-pyridinesulfonic acid, and 3-cyanopyridine. The purified enzyme exhibited an optimum pH of 8.3, and was sensitive to thiol reagents such as HgCl2 and p-chloromercuribenzoate. A reduction in the amount of cytochrome c-like component in the respiratory particles was observed during the hydroxylation reaction of nicotinic acid. Thus, nicotinic acid dehydrogenase appeared to be linked to the cytochrome respiratory chain in the cells of P. fluorescens TN5.