1. Beef-heart mitochondrial ATPase (F1) is inactivated and dissociated by incubation with 0.85 M LiCl. ATP partly protects against inactivation. Three dissociation products could be identified after chromatography on diethylaminoethylcellulose: the delta subunit which is not adsorbed, the beta subunit which may be eluted from the column, and the alpha and gamma subunits which remain bound to the column. 2. Aurovertin binds to dissociated F1 with a fluorescence enhancement equal to about 30% that found with F1. Unlike intact F1 which shows two kinetically separated phases of fluorescence enhancement, only a fast phase is found with dissociated enzyme. 3. Fluorescence measurements at varying aurovertin and protein concentrations indicate that aurovertin binds to dissociated F1 in a simple 3-component reaction with dissociation constant 0.4 muM. There are two indistinguishable binding sites, calculated on the basis of the initial F1 concentration before dissociation. 4. The beta subunit was isolated from dissociated F1 by DEAE-cellulose chromatography. It has no ATPase activity but reacts with aurovertin with a fluorescence enhancement similar to that of dissociated F1. 5. The isolated beta subunit contains one aurovertin binding site with a dissociation constant of 0.56 muM. 6. It is concluded that F1 contains two beta subunits.