Background: The microsporidian Septata intestinalis, recently suggested to be reclassified as Encephalitozoon intestinalis, is probably the second most common microsporidian isolated from AIDS patients after Enterocytozoon bieneusi. S. intestinalis causes a disseminated disease, including infections of the gastointestinal tract, whereas E. bieneusi is confined strictly to the gastrointestinal tract. It is important to differentiate between these two microsporidians, as only infections caused by S. intestinalis can, at this time, be effectively treated. Currently, diagnosis of infections caused by S. intestinalis can be achieved only by transmission electron microscopy. Methods and Results: In this study are described specific polymerase chain reaction primers for diagnosis of S. intestinalis infections based on the region coding for the small subunit ribosomal RNA cloned from a S. intestinalis isolate. These primers were tested for specificity on cloned ribosomal RNA sequences of different species of microsporidia, as well as on cultured samples of E. bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem and Vittaforma corneae (Nosema corneum), without showing any cross-amplification. By use of these polymerase chain reaction primers, eight different microsporidian isolates grown in culture and one diagnostic sample, collected as an ethanol-fixed duodenal-jejunal segment, were identified as S. intestinalis. Conclusion: These primers are powerful diagnostic tools and can enhance or replace traditional methods used to diagnose this microsporidian.