Sequence data of the small subunit rRNA (SSU-rRNA) gene were used to identify Septata intestinalis in biopsies of human immunodeficiency virus-infected patients by polymerase chain reaction (PCR), southern blot hybridization, cloning, and comparative genetic sequencing. DNA products of correct size could be amplified from all examined tissues with S. intestinalis infection but also from 2 biopsies with Enterocytozoon bieneusi and from 1 biopsy with Encephalitozoon cuniculi infection. Southern blot hybridization with an S. intestinalis-specific probe and partial sequencing of the DNA fragments showed high homology with published S. intestinalis sequences and confirmed that the amplified PCR products really derived from the SSU-rRNA gene of S. intestinalis. PCR testing can detect very light infections with S. intestinalis. Thus, S. intestinalis seems to occur more frequently in the form of latent infections and the true prevalence of the parasites may be much higher than previously reported.