Asymmetry amongst nucleotide binding sites of Escherichia coli $F_1$-ATPase was examined using $^{19}F$ NMR signal from fluorinated analogs of adenine nucleotides bound to nucleotide binding sites. ADP-$CF_2-{PO_3}^{2-}$ showed no inhibitory effect to $F_1$-ATPase. But ADP-CHF-${PO_3}^{2-}$ (racemic mixture) showed competitive inhibition of $F_1$-ATPase with $K_i$ of $60;{mu}m$. ADP-CHF-${PO_3}^{2-}$ shows only negligible binding to $EF_1$ in the absence of $Mg^2+$. With the addition of $Mg^2+$ to the medium, the $^{19}F$ resonance of free ADP-CHF-${PO_3}^{2-}$ disappeared and the new broad resonances appeared. Appearance of more than two new asymmetric resonances following the binding of ADP-CHF-${PO_3}^{2-}$ to $EF_1$ may indicate that at least one of the isomers showed split resonances. This may suggest that the region between ${alpha}$-and ${eta}$-phosphate of ADP-CHF-${PO_3}^{2-}$ which is bound to catalytic sites is experiencing a different environment at different sites.