Transhydrogenase couples reversible hydride transfer from NADH to NADP+ to proton translocation across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. The enzyme is composed of three parts. Domain I (dI) and domain III (dIII) are water soluble and contain the binding sites for NAD(H) and NADP(H), respectively; domain II (dII) spans the membrane. In the present investigation, dI from Rhodospirillum rubrum (rrI) and Escherichia coli (ecI), and dIII from R. rubrum (rrIII) and E. coli (ecIII) were overexpressed in E. coli and subsequently purified. Also, a preparation of a partially degraded E. coli transhydrogenase (ecbeta) was examined. Catalytic activities were analyzed in various dI+dIII and dI+ecbeta combinations. The abilities of the different dI+dIII combinations to catalyze cyclic transhydrogenation, i.e., the reduction of AcPyAD+ by NADH mediated via tightly bound NADP(H) in dIII, varied in the order: rrI+ecIII approximately rrI+rrIII > rrI+ecbeta >> ecI+ecIII; no measurable activities for ecI+rrIII and ecI+ecbeta were detected. Thus, rrI has a much greater apparent affinity than ecI for ecIII or rrIII or ecbeta. The pH dependences of the cyclic reaction seem to be determined by scalar protonation events on dI, both in rrI+rrIII and ecI+ecIII mixtures as well as in the wild-type R. rubrum and possibly in the E. coli enzyme. Higher reverse activities for rrI+ecbeta than for rrI+ecIII confirmed the regulatory role of dII for the association and dissociation rates of NADP(H).