Naegleria fowleri, a free-living ameboflagellate, is the causative agent of primary amebic meningoencephalitis. Healthy humans sporadically become infected with N. fowleri and develop fatal PAM after recreational or work exposure to freshwater; accordingly, there is a need for monitoring the presence of pathogenic amebeflagellates in public freshwater. The present study was conducted to determine whether a nested PCR assay could be used for detection of N. fowleri in freshwater habitats. PCR analysis was used to test samples from Virginia, Connecticut, Arizona, and Oklahoma for the presence of N. fowleri in lakes, ponds, soil, and domestic water supplies. The amebae were identified in all 4 states from soil and water sources, including domestic water supplies. In addition to identification in the environment, it is also important to determine virulence factors of the ameba. Although virulence factors have not been defined, resistance to complement lysis and production of phospholipases may account for pathogenicity of this ameba. Studies were performed to determine the gene encoding a complement regulatory protein, CD59, found in membrane fractions of N. fowleri. The genome of this organism has not been sequenced, therefore, we have constructed a genomic DNA library to search for putative virulence factors or drug targets. We have performed partial sequencing of 155 plasmids and have identified putative genes for cell motility, chromosome segregation, gene regulation, protein synthesis and degradation, protein regulation, cell signaling, respiration and energy production, membrane synthesis and metabolism, amino acid synthesis, as well as genes with unknown functions. Also, we have identified a putative virulence factor, a patatin-like protein. Patatin has been shown to exhibit phospholipase A2 activity in other organisms and has been shown to be involved in invasion into human tissue in certain pathogens. Northern analysis demonstrated hybridization with N. fowleri RNA at 3kb, but not with RNA from other free-living amebae tested. RT-PCR analysis was positive for pathogenic N. fowleri and negative for nonpathogenic Naegleria spp. Further studies are needed to determine whether the patatin-like protein in N. fowleri serves as a virulence factor and plays a role in invasion in human tissue.