Proton-translocating FF-ATP synthase (F-type ATPase, F-ATPase or FF) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FF enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used site-directed mutagenesis to investigate the role of amino acid residues β139, β158, β189, and β319 of E. coli FF-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FF-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The βN158L substitution produced no effect on the enzyme activity, while substitutions βF139Y, βF189L, and βV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations βF189L and βV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues β139, β189, and β319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate.