One binding site for phosphate (Pj) has been demonstrated on the reconstitutively active β subunit that has been removed from the Rhodospirillum rubrum membrane-bound ATP synthase (RrF0-F1. Under optimal conditions 1 mol of Pi is bound per mole of β subunit with a Kd of 270 ± 30 μM and a half-time of 15 min. Pi binding to β is absolutely dependent on MgCl2, and for its stable binding, MgCl2 must be present not only during the binding step but also during the elution-centrifugation step used to separate the bound and free [32P]Pj. The binding of Pj is inhibited by the presence of ATP or ADP. When present at low concentrations (5-50 μM) both nucleotides inhibit Pi binding to β in a noncompetitive manner with a Ki of 10 μM. At higher concentrations (0.1-10 mM) the inhibition becomes competitive with ATP being a much more effective inhibitor (Ki = 350 μM) than ADP (Ki = 10 mM). These results indicate that Pi binds to the MgCl2-dependent low-affinity nucleotide binding site that has been demonstrated on the isolated R. rubrum β subunit [Gromet-Elhanan, Z., & Khananshvili, D. (1984) Biochemistry 23, 1022-1028] probably at the site occupied by the γ-phosphoryl group of ATP. The observation that estimated Kd values for binding of Pi, ADP, and ATP to this MgCl2-dependent low-affinity binding site on β are very similar to the reported Km for ATP hydrolysis and for Pi and ADP during photophosphorylation indicates that this site might be the catalytic site of the RrF0-F1 ATP synthase.