Penicillium herquei IFO 4674 is a filamentous fungus that produces a large amount of hydrolases for fibrous polysaccharides. We purified two beta-xylosidases, S1 and S2. The molecular masses of S1 and S2 determined by MALDI-TOF-MS were 103,700 and 37,460 Da. The optimum pHs of S1 and S2 were 4.0 and 6.5, respectively. By several kinds of alcohols, especially glycerol, S1 was activated while S2 was unaffected or inhibited. S1 had a transxylosylation activity, while S2 did not. The s2 gene encoding xylosidase S2 was cloned by PCR with primers designed on the basis of partial amino acid sequences of S2. The s2 consisted of 1005 bp encoding 335 amino acids (37,433 Da) and had no secretion signal sequence. The deduced amino acid sequence shows a high identity to that of Bacteroides ovatus xylosidase/arabinosidase (56%), which is a member of the family 43 glycoside hydrolase.