Dientamoeba fragilis, a common intestinal protozoan parasite in Canada, has been associated with diarrhoea and abdominal pain in some patients. Seroprevalence of this organism has not been reported previously. In the present study sera from three symptomatic patients, 12 age- and sex-matched controls, and 189 randomly selected healthy individuals (age 6 months to 19 years) were tested for antibodies against Dientamoeba fragilis by an indirect immunofluorescence (IIF) assay. All three symptomatic patients infected with Dientamoeba fragilis had positive IIF titres of 80, and all 12 matched controls had positive titres ranging 20 to 160 (geometric mean titre 48). Of the 189 healthy children, 172 (91%) were positive at a serum dilution of 1:10 or higher. The specificity of the IIF assay was reinforced by immunoblotting 20 representative serum samples against Dientamoeba fragilis. In all 17 IIF-positive serum samples, a 39 kDa protein band of Dientamoeba fragilis was identified, the same band recognized by a mouse monoclonal antibody raised in our laboratory. Findings over a five-year period indicate that Dientamoeba fra-gilis was the most common protozoan, followed closely by Giardia lamblia and more distantly by Cryptosporidium parvum. The high seropositivity of 91% for Dientamoeba fragilis compares reasonably well with serologic data obtained by IIF and reported previously for Giardia lamblia (85.6%) and Cryptosporidium parvum (86%).