The protein profile and the antigenic cross-reactivity of 18 axenic isolates of Blastocystis hominis obtained from symptomatic patients with chronic diarrhea (14 isolates) showing no evidence of parasitic etiology and from patients with acute diarrhea attributable in 2 cases to Salmonella spp. were analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble proteins showed the existence of a common profile composed of 31 bands, with molecular weights ranging between 24 and >200 kDa, and minor differences in the proteins of 149, 118, 106, 50, 48, 47, and 30 kDa. These differences allowed us to classify the strains into three related patterns (I-III). In an indirect immunofluorescence assay, all strains were serologically identical, but two related antigenic groups (1 and 2) were found in double-immunodiffusion and Western-blot studies. The isolates of protein patterns I and II belonging to antigenic group 1 were isolated from patients with chronic diarrhea, whereas the four isolates from patients with acute diarrhea were clustered in protein pattern III and in antigenic group 2. These results confirm the protein and antigenic heterogeneity of B. hominis and the existence of demes with different pathogenic roles.