Isomaltotrio-dextranase (IMTD) which is extracellularly secreted by Brevibacterium fuscum var. dextranlyticum is an exo-type enzyme releasing isomaltotriose from dextran. Some properties of IMTD were reinvestigated for analyzing the gene encoding this enzyme. IMTD production was 3.7 times higher under the improved culture conditions, and was purified by ammonium sulfate fractionation, and chromatography on DEAE-Sepharose CL-6B (1st and 2nd), Bio-Gel P-100, and hydroxyapatite columns to homogeneous on SDS-PAGE. The molecular weight of the enzyme was estimated to be 70, 000 Da by SDS-PAGE. The optimum pH was 7.5, and was stable in the range of pH 5.5-9.0. The K_m value for dextran was 0.26 mg/ml, and the V value was 59.6, umol of isomaltotriose /min/mg of protein. The purified enzyme produced isomaltotriose as the sole product at the early reaction stage. Amino acid composition and N-terminal sequence were also analyzed, showing (N) -Asn-Pro-Thr-Lys-Pro-Val-Glu-Asp-Ala-.