The microsporidian species Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis were compared by using sequence data of their rRNA gene segments, which were amplified by polymerized chain reaction and directly sequenced. The forward primer 530f (5'-GTGCCATCCAGCCGCGG-3') was in the small subunit rRNA (SSU-rRNA) and the reverse primer 580r (5'-GGTCCGTGTTTCAAGACGG-3') was in the large subunit rRNA (LSU-rRNA). We have utilized these sequence data, the published data on Encephalitozoon cuniculi and Encephalitozoon hellem and our cloned SSU-rRNA genes from E. bieneusi and S. intestinalis to develop a phylogenetic tree for the microsporidia involved in human infection. The higher sequence similarities demonstrated between S. intestinalis and E. cuniculi support the placement of S. intestinalis in the family Encephalitozoonidae. This method of polymerized chain reaction rRNA phylogeny allows the establishment of phylogenetic relationships on limiting material where culture and electron microscopy are difficult or impossible and can be applied to archival material to expand the molecular phylogenetic analysis of the phylum Microspora. In addition, the highly variable region (E. coli numbering 590-650) and intergenic spacer regions in the microsporidia were noted to have structural correspondence, suggesting the possibility that they are coevolving.