A sensitive enzyme-linked immunosorbent assay is described for paracoccidioidomycosis serodiagnosis, with antigen represented by filtrates from Paracoccidioides brasiliensis cultures. Cross-reactivities were, however, observed with sera from patients with other mycoses such as histoplasmosis, lobomycosis, cryptococcosis, candidiasis, and sporotrichosis. These cross-reactions did not occur when we used as antigen Yarzabal E2 component prepared by affinity fractionation of the culture filtrates. Specific results could also be obtained with the whole filtrate antigen when sera were absorbed with Histoplasma capsulatum yeast and mycelial components. Besides a very high sensitivity (100% of the 69 cases of paracoccidioidomycosis tested), this assay showed no false-positive results for the 206 non-paracoccidioidomycosis sera studied, including those from patients with other mycotic diseases.