The hydrogen-deuterium exchange reaction catalyzed by the soluble hydrogenase (hydrogen:NAD + oxidoreductase, EC 1.12.1.2), an iron-sulphur flavoprotein form Alcaligenes eutrophus HI6, has been measured using H 2 gas and 2H 2O buffer. In agreement with previous studies on other hydrogenases (Gitlitz, P.H. and Krasna, A.I. (1975) Biochemistry 14, 2561–2568; Yagi, T., Honya, M. and Tamiya, N. (1968) Biochim. Biophys. Acta 153, 699–705) we have found the H/ 2H-exchange to be clearly dependent on the p 2H value. The p 2H maxima for H 2H and 2H 2 formation were determined to be at p 2H 6.2 and 6.5, respectively. The p 2H maximum of the NAD reduction is at 8.85. Resembling the reduction of NAD and of other acceptors, the aerobically purified enzyme needs to be converted to an active form for catalysis of the H/ 2H-exchange reaction. Autocatalytic activation of the free hydrogenase is observed after lag-phases the lengths of which depend on the enzyme concentration. However, comparative experiments show that autocatalytic activation does not occur with immobilized hydrogenase. This is explained by the following two steps. (i) An active hydrogenase molecule is reduced by hydrogen. (ii) Electrons are intermolecularly transferred from such an activated and reduced molecule to a nonactivated enzyme molecule. This step results in two activated molecules. NAD(P)H immediately activates the enzyme to catalyze the exchange reaction, whereas with 2-mercaptoethanol a lag-phase is observed. During the reduction of acceptors such as NAD, methyl viologen and 2,6-dichlorophenol-indophenol, the H/ 2H exchange is found to be partially suppressed. The 2H 2/H 2H ratio is p 2H-dependent: maximum value 0.38 at p 2H 8.2. During the reduction of acceptors it gradually increases or decreases depending on the type of acceptor. In the absence of any acceptor, a constant ratio of 0.36 and 0.33 is observed with NAD(P)H-activated immobilized and free hydrogenase at p 2H 8.0, respectively. From this rather small difference we assume that the 2H 2/H 2H ratio of free hydrogenase is not influenced by the enzyme concentration, since the immobilized state represents an enzyme density which is not possible with free enzyme (0.0017 mg/ml vs. 25.0 mg/ml void volume of the of glass carrier).