The chemical nature of the phosphate bond in the enzyme-P (the enzyme-phosphate complex), the activated intermediate of ATP citrate lyase [EC 4.1.3.8], was investigated. The linkage between the enzyme and phosphate in the complex was more labile in acid than in alkali. The pH stability curve of the enzyme-P is very similar to that of aspartyl phosphate and differs from that of butyl thiophosphate. The phosphorylated form of the enzyme was digested with pronase and the phosphopeptides were isolated by paper electrophoresis. When the phosphopeptides were treated with hydroxylamine, bound phosphates were liberated as P1 with concomitant formation of peptidylhydroxamates. After conversion of the peptidylhydroxamates to the dinitrophenylhydroxamate derivatives, Lossen rearrangement of the latter and the subsequent hydrolysis yieldedα, γ-diaminobutyric acid, indicating the presence of glutamyl-γ-phosphate in the hydroxylamine-sensitive groups of the original protein. From these results, it is concluded that the binding site of phosphate in the phosphorylated intermediate of ATP citrate lyase is the γ-carboxyl groups of glutamic acid residues.