A real-time polymerase chain reaction (PCR) method targeting the 5.8S ribosomal RNA gene was developed for the detection of Dientamoeba fragilis in stool samples. The PCR also included an internal control to detect inhibition of the amplification by fecal constituents in the sample. The assay was performed on species-specific DNA controls (n=29) and a range of stool samples (n=85), and achieved high specificity and sensitivity. D. fragilis DNA could be detected in unpreserved fecal samples up to 8 weeks after storage at 4 degrees C. The use of this assay in a diagnostic laboratory offers the possibility of introducing DNA detection as a feasible technique for the routine diagnosis of intestinal D. fragilis infections.