Three monoclonal antibodies (mAbs), S4D1, S3B3 and S1E5, were produced from hybridoma cell lines raised from mice immunized with hyphal walls of Neurospora crassa and one (Pax-1) from mice immunized with hyphal walls of Paxillus involutus. In immunofluorescence studies, the three N. crassa mAbs recognized epitopes with different patterns of distribution at the hyphal surface of N. crassa. S4D1 recognized an epitope which was present on the surface of both conidia and hyphae; S3B3 recognized an epitope seen only at the ends of conidia or in the septal region of hyphae and conidial chains; and S1E5 recognized an epitope present on the surface of hyphae, but not on mature conidia. mAb Pax-1 reacted with hyphal wall fragments of Pax. involutus and with N. crassa conidia in a similar way to S3B3. S4D1 reacted with an epitope found in 1,3-alpha-glycan preparations from hyphal walls of different fungi. The surface distribution of this epitope varied: it was found on the surface of both conidia and hyphae of N. crassa and Aspergillus nidulans, on the basidiospore surface only of Amanita muscaria, and on the hyphae but not the conidia of Penicillium chrysogenum. Immunogold studies revealed that the epitope was present throughout the wall of conidia and hyphae of N. crassa. mAbs S3B3, S1E5 and Pax-1 also reacted with other fungi: for example Pax-1 cross-reacted with all fungi tested except for a member of the Zygomycota. Immunogold studies revealed that epitopes of these three mAbs were present within the inner layers of the walls of conidia and hyphae of N. crassa.