Two hundred and fifty microorganisms were tested for their ability to produce β-D-xylopyranosidase. Two of the best, Aspergillus niger and Penicillium wortmanni, were studied in greater detail for the effects of inducing compounds and of surfactants on yields. Under optimal conditions, culture filtrates were obtained which had specific activity values 10 to 60 times those of sources (sea snail, Bacillus, Chaetomium, hemicellulase) used by other investigators. In most instances, our unpurified filtrates had higher activities than did the extensively purified preparations from these other sources. The fungal β-D-xylopyranosidases hydrolyze aryl and alkyl β-D-xylopyranosides, oligomers of D-xylose, and L-serine β-D-xyloside. They do not hydrolyze β-D-xylosyl dextran, tomatin, or α-D-xylosides.