In order to distinguish the pathways involved in the oxidation of matrix NADH in plant mitochondria, the oxidation of NADH and nicotinamide hypoxanthine dinucleotide (reduced form) was investigated in submitochondrial particles prepared from beetroot (Beta vulgaris L. cv. Derwent Globe) and soybeans (Glycine max L. cv. Bragg). Nicotinamide-hypoxanthine-dinucleotide(reduced form)-oxidase activity was more strongly inhibited by rotenone than the NADH-oxidase activity but both of the rotenone-inhibited activities could be stimulated by adding ubiquinone-1. The corresponding ubiquinone-1-reductase activities were inhibited by rotenone (to 69%) and further inhibited by N,N'-dicyclohexylcarbodiimide (to 79%), whilst the K3Fe(CN)6-reductase activities were not sensitive to either rotenone or N,N'-dicyclohexylcarbodiimide. Immunological analysis of mitochondrial proteins using an antiserum raised against purified beetroot complex I indicated very few differences between soybean and fresh and aged beetroot mitochondria, despite their varying sensitivities to rotenone. We confirm that there are two dehydrogenases capable of oxidising internal NADH and that only one of these, namely complex I, is inhibited by rotenone. Further, we conclude that complex I has two potential sites of quinone reduction, both sensitive to N,N'-dicyclohexycarbodiimide inhibition but only one of which is sensitive to rotenone inhibition.