Nfa1 protein expressed by the nfa1 gene that was cloned recently from pathogenic Naegleria fowleri was found in pseudopodia, especially food-cups, and concerned with a mechanism of pathogenicity of N. fowleri. In the present study, N. fowleri nfa1 gene was knocked down using double-stranded RNAs, and the expression of Nfa1 protein was observed. Using synthetic double-stranded RNA of the nfa1 gene in vitro, the nfa1 gene and Nfa1 protein were knocked down about 50.4+/-3.1% and 52+/-2%, respectively. These results suggest that RNA interference (RNAi) may be an effective technique for gene knock-down in N. fowleri trophozoites.