A method for the isolation and purification of a reversible NADH‐NADP+ transhydrogenase from Azotobacter vinelandii is described. The purification consists of 7 steps: preparation of cell‐free extract, heat treatment, first ammonium‐sulfate fractionation, calcium‐phosphate‐gel fractionation, second ammonium‐sulfate fractionation, first and second differential centrifugation and solubilisation in the presence of NADP+. The purification procedure is hampered by association‐dissociation phenomena, resulting in large losses of transhydrogenase activity. The possibility of the existence of a multi‐enzyme complex is discussed. The enzyme is purified 700–800 fold, with a yield of 15%. It catalyses a reversible hydrogen transfer between NADH and NADP+; it is able to reduce the thio‐analogues of both pyridine nucleotides, 2,6‐dichlorophenol indophenol and potassium ferricyanide with both NADH and NADPH. The activities with NADPH are on the average higher than with NADH. The reduction of oxidised thionicotinamide‐adenine dinucleotide (phosphate) by NADH is only influenced by adenosine 2′‐monophosphate in phosphate buffer at higher pH values. The purified transhydrogenase is a flavoprotein with FAD as prosthetic group; the absorption spectrum of the NADP+‐free enzyme is characterised by a maximum at 442 nm. A minimum molecular weight of 60000 can be calculated from the experimentally determined absorption coefficient. ɛ442 nm = 12900 M−1cm−1. The oxidised enzyme is stable at elevated temperatures and at high dilution; storage at –20°C results in aggregation of the enzyme. The reduced enzyme is thermolabile; inactivation at elevated temperatures can be prevented by the addition of FAD. Preparation of the apoenzyme by different procedures is difficult and incubation with FAD only results in partial restoration of the transhydrogenase activity. Ultracentrifugation studies demonstrate the presence of three components with sedimentation coefficients (s20,w) of 24, 48 and 88 S. In the presence of NADPH and NADP+ a relation between the 48‐ and 88‐S components can be demonstrated. The sedimentation coefficient of the main component is concentration independent beyond 1 mg/ml; at lower concentrations a tendency to higher sedimentation values is obtained. From light‐scattering experiments a molecular weight (Mr) of 30–50 millions is determined; the calculated length for a rod‐like structure is 1000–1500 nm.