An a-galactosidase-producing fungus was screened out of 26 filamentous fungi isolated from soil by us. Phylogenetic analysis based on the alignment of 18S rDNA sequences, combined with the morphological identification, indicated that the strain F63 was a member of the genus Penicillium. The a-galactosidase from Penicillium sp. F63 was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. The molecular size of the purified enzyme is approximately 82kDa estimated by SDS-PAGE. The a-galactosidase has an optimum pH of 5.0 and an optimum temperature of 45 degrees C. The enzyme is stable between pH5.0 and 6.0 below 40 degrees C. The a-galactosidase activity is slightly inhibited by Ag+ , which is dissimilar to other a-galactosidases. Kinetic studies of the a-galactosidase showed that the Km and the Vmax for pNPG are 1.4mmol/L and 1.556mmol/L. min(-1) x mg- 1, respectively. The enzyme is able to degrade natural substrates such as melibiose, raffinose and stachyose but not galactose-containing polysaccharides. The alpha-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The results show that the a-galactosidase is a novel one.