2,4,3',5'-tetrahydroxystilbene (THS) wa s found to inhibit rat liver mitochondrial adenosine triphosphatase (ATPase) activity induced by various concentrations of 2,4-dinitrophenol (DNP). The I50 was found to be 17 nmoles/mg mitochondrial protein. The maximum inhibitory effects of oligomycin and atractyloside on the DNP-activated mitochondrial ATPase activity can be enhanced by adding THS. The atractyloside-insensitive ATPase activity of Lubrol-treated rat liver mitochondria was also inhibited by low concentration of THS. The tetramethoxyderivative of THS was much less effective than the parent compound in depressing the ATPase activity of both intact and Lubrol-treated mitochondria. These observations suggest that the phenolic groups are essential for the mitochondrial actions of THS, and this compound most probably acts by a mechanism different from oligomycin on the mitochondrial ATPase complex.