One of the processes by which CheY, the excitatory response regulator of chemotaxis in Escherichia coli, can be activated to generate clockwise flagellar rotation is by acetyl-CoA synthetase (Acs)-mediated acetylation. Deletion of Acs results in defective chemotaxis, indicating the involvement of Acs-mediated acetylation in chemotaxis. To investigate whether Acs is the sole acetylating agent of CheY, we purified the latter from a delta acs mutant. Mass spectrometry analysis revealed that this protein is partially acetylated in spite of the absence of Acs, suggesting that CheY can be post-translationally acetylated in vivo by additional means. Using [14C]AcCoA in the absence of Acs, we demonstrated that one of these means is autoacetylation, with AcCoA serving as an acetyl donor and with a rate similar to that of Acs-mediated acetylation. Biochemical characterization of autoacetylated CheY and mass spectrometry analysis of its tryptic digests revealed that its acetylated lysine residues are those found in CheY acetylated by Acs, but the acetylation-level distribution among the acetylation sites was different. Like CheY acetylated by Acs, autoacetylated CheY could be deacetylated by Acs. Also similarly to the case of Acs-mediated acetylation, the phosphodonors of CheY, CheA and acetyl phosphate, each inhibited the autoacetylation of CheY, whereas the phosphatase of CheY, CheZ, enhanced it. A reduced AcCoA level interfered with chemotaxis to repellents, suggesting that CheY autoacetylation may be involved in chemotaxis of E. coli. Interestingly, this interference was restricted to repellent addition and was not observed with attractant removal, thus endorsing our earlier suggestion that the signaling pathway triggered by repellent addition is not identical to that triggered by attractant removal.