An acidic isocitrate lyase was induced in Acinetobacter calcoaceticus grown on malonate as a sole source of carbon. This enzyme was purified 30 fold by the combined methods of ammonium sulfate fractionation, oxalate $omega$-aminohexyl agarose chromatography, DEAE-Sephacel anion exchange chromatography, Sephadex G-150 gel filtration and itaconate $omega$-aminohexyl agarose chromatography in an electrophoretically homogeneous form. Molecular size of the enzyme was estimated by Sepharose 6B gel filtration to be 129,000 dalton composed of two 64,000 dalton subunits. The optimum pH for this enzyme was 6.1 and the optimum temperature was about $40^{circ}C$. The enzyme showed a typical Michaelis-Menten kinetics for the substrate, isocitrate, from which Km was calculated to be $8.3{ imes}10^{-5}M$. The enzyme activity was inhibited uncompetitively by succinate and itaconate, and competitively by oxalate and glycolate, respectively. The enzyme activity was also inhibited strongly by N-ethylmaleimide, p-chloromercuripheny-lsulfonic acid and iodoacetamide, suggesting that the enzyme has an active -SH group on or near the active site. And diethylpyrocarbonate was also an inhibitor for the enzyme activity, indicating the possible involvement of imidazole group. The purified enzyme was immunogenic in rat. The antiserum prepared against the enzyme inhibited the enzyme activity more than eighty percent.