Paracoccidioidomycosis (PCM) is a chronic granulomatous infectious disease, endemic in subtropical areas of Central and South America. The diagnosis of the central nervous system (CNS) involvement with PCM (neuroparacoccidioidomycosis [NPCM]) frequently is difficult. A definitive diagnosis usually is made by visualization or isolation of Paracoccidioides brasiliensis from CNS biopsy or necropsy material. In the present study, we determined the presence of anti-gp43 antibodies in the cerebrospinalfluid (CSF) of patients with CNS involvement in PCM by enzyme-linked immunosorbent assay (ELISA) in 9 cases of NPCM and 15 control cases. ELISA anti-gp43 was compared with double immunodiffusion (DID). ELISA anti-gp43 was positive in 8 (89%) of 9 CSF samples from patients with NPCM and negative in all CSF samples of the control group. DID was negative in all CSF samples from patients with NPCM and control samples. ELISA anti-gp43 in CSF samples is better than DID for the diagnosis of NPCM. It is a sensitive and specific diagnostic method and has high predictive values. To our knowledge, this is thefirst time ELISA anti-gp43 was applied to CSF.