Acanthamoeba is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of Acanthamoeba are medically relevant as the causative agent of sight-threatening Acanthamoeba keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. Previous work employing DNA sequences of nuclear and mitochondrial small-subunit rRNA genes (SSU rRNA genes) determined the genotypic diversity of Acanthamoeba and found that many named species of Acanthamoeba are associated with particular genotypes. These studies also concluded that nearly all AK infections result from a single molecular genotype: T4. Here, we asked whether Acanthamoeba clinical isolates from non-AK infections are also associated with particular genotypes. DNA sequence determination of nuclear SSU rRNA genes was employed for genotypic identification of 29 isolates of Acanthamoeba from non-AK infections. Sequence analysis demonstrates that T4 is the predominant genotype in non-AK infections, including those in brain, cerebrospinal fluid, nasal passages, skin, and lung. Rare genotypes (T1, T10, and T12) have been isolated from brain infections. We conclude that genotype T4 is the primary genotype in non-AK Acanthamoeba infections, as was the case in AK infections. However, the genotypes that were isolated from brains have not been observed in environmental isolates of Acanthamoeba, and their natural ecological niche is unknown.The genus Acanthamoeba is a group of nearly 25 named species that has a worldwide distribution. Members of Acanthamoeba are naked (i.e., lacking tests, walls, or tecta with openings), free-living amebae that inhabit a wide range of natural ecological niches, including fresh and brackish water, beach sand, soil, air, etc. (4, 13, 16, 17). Acanthamoeba also has been isolated from humans and animals (16,18). In addition to its natural distribution, Acanthamoeba can be opportunistically pathogenic, being identified as the causative agent of a painful and sight-threatening infection of the cornea, Acanthamoeba keratitis (AK) (22). These infections can occur in otherwise healthy, nonimmunocompromised individuals. In developed countries, AK infections are usually associated with contact lens wear. Improper sterilization procedures have been identified as the cause of a large percentage of such AK infections (2, 22, 25). In developing nations, AK infections are not generally associated with contact lens wear; by contrast, most cases are the result of ocular trauma (6, 23).Acanthamoeba is also responsible for life threatening infections in patients with immunodeficiency disease. These include documented infections of the skin, nasal passages, lung, and brain (16,17,18,19). In the brain, Acanthamoeba causes a severe encephalitis termed granulomatous amebic encephalitis, which is nearly always fatal (18). Variations in the pathogenicity of different Acanthamoeba strains have been recognized in laboratory studies, but the relevance of these results to hum...