F1F0-ATP synthase was purified from Escherichia coli βY331W mutant. The β-Trp-331 provided a specific fluorescent probe of catalytic site nucleotide binding. Physiological (mM) concentration of substrate MgATP filled all three catalytic sites. With MgATP or MgADP the catalytic sites showed marked binding cooperativity and asymmetry, which was dependent on Mg2+, Nucleotide binding was fast, with k(on) = ~6 x 105 M-1 s-1. P(i) at physiological concentration (5 mM) did not bind to catalytic sites. Measurement of MgATP hydrolysis and binding under identical conditions as a function of MgATP concentration revealed that V(max) was achieved only when all three catalytic sites were filled in every enzyme molecule. The enzyme species with two catalytic sites occupied and one site empty displayed low, nonphysiological catalytic rate. This is the first characterization of nucleotide binding parameters in F1F0. The fact that the behavior of purified F1F0 was similar in most respects to that of isolated F1 demonstrated that the presence of the additional F0 subunits a, b, and c, and also fixed stoichiometric amounts of ε and δ, does not affect catalytic site properties. The results impact on possible catalytic mechanisms, namely, they emphasize that P(i) cannot simply bind spontaneously, that an enzyme species with all three sites occupied is the only catalytically competent species, and that release of product and binding of substrate cannot lie simultaneous, rather the former must precede the latter.