It had previously been suggested that V(max) hydrolysis rate of 2',3'-O- (2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that V(max) rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using βY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at V(max) rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, 'bisite hydrolysis' of MgTNP-ATP amounted to a substantial fraction (~40%) of V(max). Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (K(d1) < 1 nM, K(d2) = 23 nM, K(d3) = 1.4 μM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1- ATPases anti that these three properties are strongly correlated.