Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate. In contrast to citrate lyase ligase from C. sporosphaeroides and Streptococcus lactis, the enzyme from C. sphenoides was under stringent regulatory control. The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase. These enzymes were purified from in vivo32P-labeled C. sphenoides cells, which were grown on low-phosphate medium containing 40 mM citrate and 1 mCi [32P]orthophosphate. During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled. Growth experiments with 14C-labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide. Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated. Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation.