SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli. In this study, we investigated the cleavage specificity of Lon protease toward SulA protein. The enzyme was shown to cleave approximately 27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P1, and P1' positions, respectively, and one or two Gln residues in positions P2-P5. They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively. The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP. The central region was predicted to be rich in alpha helix and beta sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease.