We tested 513 stool samples from patients in Sydney, Australia for Blastocystis by using five diagnostic techniques: microscopy of a permanently stained smear using a modified iron-hematoxylin stain, two xenic culture systems (a modified Boeck and Drbohlav's medium and tryptone, yeast extract, glucose, methionine-9 medium), and two published conventional polymerase chain reaction methods specific for the small subunit ribosomal DNA. Ninety-eight (19%) samples were positive for Blastocystis in one or more of the diagnostic techniques. The PCR 2 method was the most sensitive at detecting Blastocystis with a sensitivity of 94%, and the least sensitive was microscopy of the permanent stain (48%). Subtype 3 was the most predominant subtype (present in 43% of samples assigned to this group). This study highlights the low sensitivity of microscopy when used as the sole diagnostic modality for detection of Blastocystis sp.