The isolated β subunit of the Rhodospirillum rubrum F0.F1 ATP synthase contains two nucleotide binding sites, a Mg-independent and a Mg-dependent site [Gromet-Elhanan, Z., & Khananshvili, D. (1984) Biochemistry 23, 1022–1028]. Phosphate (Pi) binds only to the second site [Khananshvili, D., & Gromet-Elhanan, Z. (1985) Biochemistry 24, 2482–2487]. Binding of these ligands has been found to induce conformational changes in the β subunit that can be followed by their effect on trypsin sensitivity of the subunit. With a ratio of 1 mol of trypsin/100 mol of β, the subunit is digested in the absence of ligands with a half-time of 10 min. MgCl2has no effect on the trypsin sensitivity of β, but the other ligands show pronounced effects. Binding of either ADP or ATP to the Mg-independent site results in partial protection of the β subunit against its digestion by trypsin, increasing the t1/2 to 20 min. A further decrease in the sensitivity to trypsin occurs on binding of Mg ADP to the second Mg-dependent site, increasing the t1/2 to 30 min. Binding of MgATP or MgPi, to this site causes, however, an opposite effect, resulting in a decrease in the t1/2to 3 and 6 min, respectively. These results indicate that ligand binding induces two distinct changes in the conformation of the isolated β subunit. One conformational state is obtained on occupation of the Mg-independent nucleotide binding site and is further stabilized by MgADP. The second conformational state is obtained on binding of MgATP or MgPisuggesting that it is induced by occupation of the γ-phosphoryl subsite in the Mg-dependent catalytic site on the β subunit.