The pyruvate-aspartic β-semialdehyde condensing enzyme was purified about 1,000-fold from cell-free extracts of Bacillus subtilis PCI-219. The molecular weight of the condensing enzyme was determined to be 124,000±2,000 by gel filtration on a Sephadex G-200 column. The enzyme exhibited a sharp pH optimum at pH 9.5 in glycine-NaOH buffer. The Km values for pyruvate and ASA were determined to be 1.07×10−3 and 3.13×10−3M, respectively. The enzymatic activity was inhibited by α, ε-diketopimelic acid and glyoxylic acid and was activated by isocitric acid. Double-reciprocal plots showed that diketo-pimelic acid was a competitive inhibitor with respect to ASA and pyruvate, and its K1 values were determined. A comparison of the condensing enzyme from vegetative cells with that from sporulating cells was made, but no difference was observed as regards molecular weight or in the effects of diketopimelic acid, pyridine-2,6-dicarboxylic acid (dipico-linic acid), L-lysine, and diaminopimelic acid. The o-aminobenzaldehyde method for determination of 2,3-dihydropyridine-2,6-dicarboxylic acid (dihydrodipicolinic acid) was modified in order to give more satisfactory results for strict determination of the condensing enzyme activity.