Citrate lyase from Klebsiella aerogenes inactivated by reaction in the presence of substrate or by treatment with hydroxylamine can be reactivated with acetic anhydride only if its sulfhydryl groups are reduced. Alkaline hydrolysis of pure citrate lyase yields about 3 mol of phosphopantothenate per mol of enzyme. Citrate (pro-3S) lyase (EC 188.8.131.52) catalyzes the cleavage of citrate to acetate and oxaloacetate, and requires the presence of Mg2+ or certain other divalent metal ions. The enzymes from Escherichia coli and Klebsiella aerogenes have been shown by Singh and Srere (1) to undergo a "reaction-inactivation" in the presence of Mg2+, Mn2+, or C02+, but not when Zn2+ is used in the reaction. In our studies attempting to reactivate the reaction-inactivated enzyme, we found that hydroxylamine was an inhibitor for the enzyme and that acetic anhydride could reactivate the hydroxylamine-inhibited enzyme (2). Acetic anhydride gave variable results in attempts to reactivate the reaction-inactivated enzyme. Recently, Buckel et al. (3) showed that citrate lyase contained an acetyl group that is necessary for enzyme activity. These workers showed that the acetyl group on the enzyme turned over during the course of the reaction; they also showed that inactivation of the enzyme by oxaloacetate (4) was due to the deacetylation of enzyme, and suggested the reaction-inactivation phenomena was also related to this process. Buckel et al. (3) suggested that the acetyl group was present as a thioester on the enzyme. In this paper we will present further evidence that the acetyl group on citrate lyase occurs as a thioester. We will also show that stoichiometric amounts of phosphopantothenate can be isolated from alkaline hydrolysates of the enzyme.