ABSTRACT The mitochondrial, inner-membrane–associated, reversible NADPH → NAD + transhydrogenase of the energetically anaerobic adult cestode Hymenolepis diminuta connects NADPH generation, via a mitochondrial NADP + -specific “malic” enzyme, with NADH formation needed for electron transport. In reducing the pyridine nucleotide, the enzyme concomitantly catalyzes transmembrane proton translocation, thereby coupling NADH formation to ATP generation or NADPH formation to ATP hydrolysis. Detergent-solubilized transhydrogenase, from isolated mitochondrial membranes, was purified to apparent homogeneity using ion exchange and hydroxylapatite chromatographies. The enzyme displayed a monomeric M r of ∼110 kDa and required phospholipid, without which activity was rapidly lost. Of the phospholipids examined, phosphatidylcholine was the most effective. Transhydrogenase-catalyzed NADH formation was inhibited by NAD(P) + and adenylates, suggesting regulatory effects of the pyridine nucleotides and effects of pyridine nucleotide-simulating molecules. In keeping with its proton-translocating function, the enzyme was inhibited by dicyclohexylcarbodiimide. The isolated enzyme catalyzed neither NADH → NADP + nor NADH → NAD + transhydrogenations, thereby suggesting a need for a minimal coupling to electron transport for the NADH → NADP + reaction as well as enzyme specificity. Anti-transhydrogenase monospecific antibodies proved inhibitory to NADPH → NAD + transhydrogenation catalyzed by both isolated and membrane-associated enzymes. This purification study apparently represents a first for parasitic helminths or multicellular invertebrates generally and establishes a framework for evaluating the transhydrogenase as a potential site for specific chemotherapeutic attack.