An oriented expression library was constructed from the mycelia of the symbiotic model fungus Hebeloma cylindrosporum in the high-level yeast expression vector pDR196. DNA sequencing of approximately 500 expressed sequence tags (ESTs) showed that 15% correspond to known genes, two thirds contain sequences with unknown function, andthe remaining 20% showed no significant similarity to any known genes. The ESTs had a GC content between 44 and 56%, with most of them having a GC content of 52-54%, which could be correlated with GC contents of fungal genes. The library was successfully used to identify the Hebeloma HIS4 gene by functional complementation of a yeast his4 mutant. Thus, the library may serve as a powerful tool for identification and characterization of mycorrhizal genes by EST analysis and for the identification of ectomycorrhizal genes by means of suppression cloning.