Naegleria fowleri, a free-living amoeba, causes fatal primary amoebic meningoencephalitis in experimental animals and humans. The nfa1 gene (360 bp) was previously cloned from a cDNA library of pathogenic N. fowleri by immunoscreening, and produced a 13.1-kDa recombinant protein that showed pseudopodia-specific localization by immunocytochemistry. On the basis of an idea that the pseudopodia-specific Nfa1 protein seems to be involved in the pathogenicity of N. fowleri, the cytopathic activity of N. fowleri trophozoites co-cultured with rat microglial cells was observed, and the effects of an anti-Nfa1 antibody in a co-culture system were elucidated. Using light, scanning and transmission electron microscopy, it was seen that N. fowleri trophozoites in contact with microglial cells produced vigorous pseudopodia and a food-cup structure. Microglial cells were destroyed by N. fowleri trophozoites as seen from necrotic cell death in a time-dependent manner. In a(51)Cr release assay, N. fowleri showed 17.8%, 24.9%, 54.6% and 98% cytotoxicity against microglial cells at 3, 6, 12 and 24 h post-incubation, respectively. However, when anti-Nfa1 antibody was added in a coculture system, N. fowleri cytotoxicity was reduced to 15.5%, 20.3%, 46.7% and 66.9%, respectively. Moreover, microglial cells co-cultured with N. fowleri trophozoites secreted the pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6. In the presence of anti-Nfa1 antibody, the secretion of TNF-alpha was slightly, but not significantly, decreased.