The plant-pathogenic fungus Cochliobolus carbonum secretes one major beta-xylosidase (Xyp1) when grown on xylan or maize cell walls. cDNA and genomic DNA encoding Xyp1 were isolated using PCR primers based on peptide sequences from the purified protein. XYP1 contains three introns, has 5' and 3' untranslated regions of 74 and 145 bp, respectively, and is predicted to encode a protein of 328 amino acids (Mr 36700) with four N-glycosylation sites. Although it is secreted, Xyp1 has no predicted signal peptide. Furthermore, Xyp1 appears not to be processed at the N-terminus because one of the peptides isolated from the mature protein is located only six amino acids downstream of the translational start methionine. The primary sequence of Xyp1 is unrelated to any known eukaryotic beta-xylosidase but has 35% overall identity to two bacterial bifunctional beta-xylosidase/alpha-arabinosidases. Mutation of XYP1 by targeted gene replacement resulted in the loss of the major beta-xylosidase activity corresponding to the product of XYP1, but a significant amount of secreted beta-xylosidase activity (25% of wild-type) remained in the culture filtrates. The xyp1 mutant was still fully pathogenic on maize.