An exo-ß-1,3-D-galactanase (Fo/1,3Gal) was purified from the culture filtrate of Fusarium oxysporum 12S. A cDNA encoding Fo/1,3Gal was isolated by in vitro cloning. Module sequence analysis revealed a "GH43_6" domain and a "CBM35_galactosidase-like" domain in Fo/1,3Gal. The recombinant enzyme (rFo/1,3Gal) expressed in Pichia pastoris degraded ß-1,3-galactan and ß-1,3-galactobiose (Gal2), and released only galactose (Gal). In contrast, the enzyme did not hydrolyze p-nitrophenyl ß-D-galactopyranoside, ß-1,4-Gal2, or ß-1,6-Gal2. The enzyme also showed low activity towards native type II arabinogalactans such as larchwood arabinogalactan (LWAG) and gum arabic. Using LWAG as substrate, rFo/1,3Gal released Gal, ß-1,6-Gal2, ß-1,6-galactotriose (Gal3), and ß-1,6-Gal3 substituted with a single arabinofuranose residue accompanied with unidentified oligosaccharides, indicating that the enzyme can by-pass the branching points of ß-1,3-galactan backbones. A time course analysis of products released by rFo/1,3Gal on LWAG revealed that ß-1,6-Gal2 is the main side chain in LWAG and that the activity of rFo/1,3Gal was decreased when degrees of polymerization of side chains increase. rFo/1,3Gal worked synergistically with three other recombinant F. oxysporum enzymes (ß-1,6-galactanase, ß-L-arabinopyranosidase, and α-L-arabinofuranosidase) that degrade side chains, on the degradation of LWAG. However, the synergism was much lower than anticipated, probably because LWAG have longer side chains than the three enzymes used are able to remove or ß-1,3-galactan main chain is interrupted with glycosidic linkages that are different from the ß-1,3-galactosyl linkage. Affinity gel electrophoresis revealed that rFo/1,3Gal specifically bound to ß-1,3-galactan.