The environmentally resistant spores of Encephalitozoon intestinalis, E. cuniculi, and E. hellem may be acquired by ingestion of contaminated food or water. These species, along with Enterocytozoon bieneusi, may represent serious health threats for immunocompromised populations, and can also infect immune-competent individuals. E. bieneusi and E. intestinalis are both on the CCL-1 list, which supports investigation for detection and viability determination procedures. Presently, in vitro spore production is not available for E. bieneusi, but the Encephalitozoon spp. are very adaptable to cell culture production. Recent advances in molecular techniques have made it possible to both detect select microorganisms in water, and determine their viability. The polymerase chain reaction (PCR) can detect a pathogen's DNA, and therefore assist in organism identification, while the reverse-transcription (RT)-PCR technique, which detects RNA, can be used to determine an organism's viability. RK-13 cells were seeded into individual wells of 24 well plates for the RT-PCR CC assay and onto coverslips for the Microwell assay (Wolk et al., 2000). Heat-inactivated or untreated spores were then inoculated onto the 24 well plates or coverslips. The Microwell assay plates were incubated for six days, while the RT-PCR CC plates were incubated for three days post infection. Total RNA was isolated from the individual cell culture wells using the SNAP RNA Isolation Kit (Invitrogen). The GeneAmp RNA PCR Kit (Perkin Elmer) was used to convert the mRNA to cDNA prior to amplification by PCR. PCR products were detected by agarose gel electrophoresis. The kits allow all of the process to be completed in one column. The 16S rRNA gene sequences for the human pathogens E. cuniculi, E hellem, E. intestinalis, and E. bieneusi have been determined and are available in the GenBank database, and PCR primers designed to the 16S rRNA genes have been used to identify microsporidia in clinical samples (Weiss and Vossbrinck, 1999). The 16S rRNA primers evaluated in this study were SI500 (Weiss et al., 1994) and PMP1/VI (Fedorko et al.,1996). These primers were able to amplify products from untreated, or live spores, in either PCR or RT-PCR CC assays, which compares well with the in vitro Microwell plate assay. However, PCR with these primers could not be used to determine spore viability, as they were able to amplify PCR products from both live and dead spores. The second primer set evaluated was designed to amplify a region within the ¿-tubulin gene. The third primer set evaluated was designed to amplify a region within the Hsp70 gene. From the three primer sets evaluated, the ¿-tubulin set has demonstrated an equivalency comparable to the cell culture coverslips at 104 spores for E. intestinalis and E. hellem, similarly with Hsp70 set for E. hellem and E. cuniculi. In order to use a RT-PCR assay for water samples or viability testing, it may be necessary to use a nested primer approach, a multiplex approach or to develop additional primer sets that would identify all three species with one assay. The study, however, demonstrated that the RT-PCR CC assay may become a useful tool for disinfection evaluation assays. Includes 6 references, tables.