A type II arabinogalactan-degrading enzyme, termed Exo-1,3-Gal, was purified to homogeneity from the culture filtrate of Sphingomonas sp. 24T. It has an apparent molecular mass of 48 kDa by SDS-PAGE. Exo-1,3-Gal was stable from pH 3 to 10 and at temperatures up to 40 A degrees C. The optimum pH and temperature for enzyme activity were pH 6 to 7 and 50 A degrees C, respectively. Galactose was released from beta-1,3-d-galactan and beta-1,3-d-galactooligosaccharides by the action of Exo-1,3-Gal, indicating that the enzyme was an exo-beta-1,3-d-galactanase. Analysis of the reaction products of beta-1,3-galactotriose by high-performance anion-exchange chromatography revealed that the enzyme hydrolyzed the substrate in a non-processive mode. Exo-1,3-Gal bypassed the branching points of beta-1,3-galactan backbones in larch wood arabinogalactan (LWAG) to produce mainly galactose, beta-1,6-galactobiose, and unidentified oligosaccharides 1 and 2 with the molar ratios of 7:19:62:12. Oligosaccharides 1 and 2 were enzymatically determined to be beta-1,6-galactotriose and beta-1,6-galactotriose substituted with a single arabinofuranose residue, respectively. The ratio of side chains enzymatically released from LWAG was in good agreement with the postulated structure of the polysaccharide previously determined by chemical methods.