Blastocystis is a common single-celled enteric parasite found in a large variety of hosts. Recent molecular analysis supports the concept that this eukaryotic organism is a stramenopile most closely related to Proteromonas lacertae, a parasite of reptiles. In this study, the internal transcribed spacer region, partial small subunit rRNA and large subunit rRNA genes from 7 Blastocystis isolates (5 human, 1 pig and 1 sheep), and a Proteromonas lacertae isolate were amplified by PCR, cloned and sequenced. Blastocystis was found to be a typical eukaryote with both ITS1 and ITS2 regions present. Phylogenetic analysis based on the entire PCR amplicon revealed that the Blastocystis isolates did not segregate according to host or geographic origin. The highest sequence identities with the conserved Blastocystis 5·8S rDNA sequence were with the stramenopiles Fibrocapsa japonica, Chattonella marina, Cylindrotheca closterium and Hyphochytrium catenoides. The most parsimonious tree based on the 5·8S rDNA sequence from P. lacertae, 11 other stramenopiles, 2 fungi, 3 algae and 3 alveolates showed Blastocystis positioned within the stramenopiles, with P. lacertae as its closest relative. This work therefore supports the hypothesis that Blastocystis is most closely related to P. lacertae, and that it should be regarded as an unusual stramenopile.