We investigated the existence of an NADH-dependent paraquat (PQ) reduction system in rat liver mitochondria (Mt) in respect to the cytotoxic mechanisms of PQ. The outer membrane fractions, free from the contamination of inner membranes but with a few microsomes, catalyzed rotenone-insensitive NADH, but not NADPH, oxidation by menadione or PQ. Anti-NADH-cytochrome b5 reductase antibody and its inhibitor p-hydroxymercuribenzonate did not inhibit the NADH-PQ reduction activity. Therefore, the respiratory systems of the inner membranes and microsomal cytochrome P450 systems could not have been responsible for the reaction. Dicoumarol, an inhibitor of NAD(P)H-quinone oxidoreductase (NQO), dose dependently suppressed the NADH oxidation in the outer membrane via PQ as well as menadione, with I50 values of 190 (for menadione) and 150 microM (for PQ). Because of a lower sensitivity to NADPH and the higher doses of dicoumarol required for its inhibition, the activity in the outer membrane may be an "NADH-quinone oxidoreductase" which partly differs from the NQO previously reported. This outer membrane enzyme produced superoxide anions in the presence of both NADH and PQ and was too tightly membrane-bound to be extracted by Triton X-100 and deoxycholate. From these results, we concluded that the free radical-producing mitochondrial NADH-quinone oxidoreductase is a novel oxidation-reduction system participating in PQ toxicity. This is in good agreement with our previous results showing that PQ selectively damaged Mt in vivo and in vitro, resulting in cell death (K.-I. Hirai et al., 1992, Toxicology 72, 1-16).