Enzymatic analysis of oligosaccharides using exoglycosidases has become a powerful tool for determining the sequence and structure of sugar chains. The principal limitation to these methods has been the lack of highly purified and wellcharacterized enzymes. Using fluorescently labelled carbohydrate substrates and TLC, we have developed a method to identify glycosidases with novel specificities. This screening method led to the discovery that bacteria of the genus Xanthomonas are a rich source of exoglycosidases. From Xanthomonas manihotis, eight novel exoglyccosidases have been isolated and characterized. A novel β-N-acetylglucosaminidase has been purified that, unlike those previously described, will cleave N-acetylglactosamine without cleaving N-acetylgalactosamine residues. A novel β-galactosidase has been isolated that preferentially hydrolyses β(1→3) galactosyl linkages. Three α-mannosidases have been isolated that serve as useful reagents in the analysis of high-mannose oligosaccharide structures: α1–3,6 mannosidase and α1–2,3 mannosidase. An α1–3,6 galactosidase has been purified that does not hydrolyse terminal α1–4 galactose residues. Two fucosidases, α1–3,4 fucosidase and α1–2 fucosidase, are similar to enzymes purified from other sources. Together, these glycosidases provide powerful reagents for determining the sequence of complex carbohydrates. Equally important is their usefulness in selectively removing specific sugar residues and thereby creating novel carbohydrates for analysing the biological roles of oligosaccharides.