Although lacking an NADPH→NAD+ transhydrogenase system, the essentially energetically anaerobic mitochondria of the adult intestinal nematode Ascaris suum display an inner membrane-associated NADH→NAD+ transhydrogenation reaction. This reaction is considered to be reflective of a mechanism(s) that acts in catalyzing a transmembrane translocation of reducing equivalents from NADH in the intermembrane space to matrix NAD+, thereby forming matrix NADH that would serve in electron transport. Ascarid mitochondrial lipoamide dehydrogenase rather than an NADH→NAD+ transhydrogenase system has been viewed as the predominant source of inner membrane-associated NADH→NAD+ transhydrogenation activity. However, the present study made apparent yet another source of mitochondrial, inner membrane–associated NADH→NAD+ activity in A. suum, viz., NADH dehydrogenase. This was made evident via comparisons of the A. suum mitochondrial NADH→NAD+ transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities in terms of pH effects, thermal labilities, the involvement of NADH dehydrogenase in the activities of mitochondrial, membrane-associated rotenone-insensitive and rotenone-sensitive NADH-dependent cytochrome c reductases, and mitochondrial membrane versus mitochondrial soluble localizations. Studies of the responses of the NADH→NAD+ transhydrogenation, rotenone-insensitive and rotenone-sensitive cytochrome c reductases, and lipoamide dehydrogenase activities to inhibition by copper and cadmium lent additional support to the catalysis of an NADH→NAD+ transhydrogenation activity by NADH dehydrogenase. Collectively, the data presented are consistent with an additional physiological catalysis of an NADH→NAD+ transhydrogenation in A. suum mitochondria by an inner membrane NADH dehydrogenase component of the rotenone-sensitive cytochrome c reductase system, i.e., the NADH dehydrogenase component of the electron transport system. Comparisons of the A. suum data with those from other essentially anaerobic helminth parasites as well as free-living eukaryotic mitochondrial systems are noted.