The major citrate synthase of Bacillus subtilis (CS-II) was purified to near homogeneity and shown to correspond to the product of the citZ gene. Accumulation of CS-II during exponential growth and stationary phases paralleled expression of the citZ gene. The physical and kinetic properties of CS-II were similar to those of citrate synthase enzymes from Bacillus megaterium and from eukaryotic cells but differed from those of citrate synthases from many gram-negative bacteria.