Abstract Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2′-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2′-AMP, NADP +, or NADPH but not with 5′-AMP, NAD +, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2′-AMP or NADP +. With 2′-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6–1.7 million, and a Stokes' radius of 11.7 nm were determined. In the presence of NADP + the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.