The influence of the ε-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (β-Trp-331). The interaction between ε and F1 was not affected by the mutation. K(d) for binding of ε to βY331W mutant F1 was ~1 nM, and ε inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the ε-depleted and ε-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. K(d1)(MgATP) and K(d1)(MgADP) were an order of magnitude higher in the absence of ε than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the ε-depleted and ε- replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of ε, K(m) equals K(d3). Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (V(max)) MgATP hydrolysis rates.