Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD+/NADH, and domain III for NADP+/NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP+ (k approximately 0.03 s(-1)). Phase A of the burst (k approximately 600 s(-1)) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k approximately 10-50 s(-1)), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcPdAD+ reduction by [4B-2H]NADPH, and by [4B-1H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27535-27538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I:III complex.