The fungus-growing attine ants are a unique monophyletic new-world lineage comprising about 10 genera and > 200 species. With one exception all fungi living in this symbiosis are of the family Lepiotaceae (Basidiomycota: Agaricales). The most derived genera within this group, the Atta and Acromyrmex leaf-cutting ants, have realized the key innovation that they use fresh leaves as substrate for their fungus gardens, which allowed them to tap into tremendously abundant resources and to evolve enormous societies. It is generally assumed that one of the main benefits for the ants in the symbiosis is to profit from the enzymatic capacity of the fungus to degrade polysaccharides originating from plant cell walls. These polysaccharides include cellulose and hemicellulose, the two most abundant organic compounds found in nature. A key enzyme in the degradation of hemicellulose is xylanase, which is able to degrade the xylan part of hemicellulose. Using degenerate primers designed from known xylanase genes, we have PCR amplified a gene with high similarity to known xylanases, using DNA extracted from the fungal symbiont of Acromyrmex echinatior. By heterologous expression in yeast we have shown that the protein product of the gene exhibits xylanase activity. Furthermore, our results also suggest that xylanase production is not uniform within the fungus garden, but may be restricted to the lower, oldest part of the fungus garden. This suggests that the degradation of leaf material in the fungus garden takes place as a succession of enzymatic steps, ending with the degradation of the cell wall polysaccharides.