Microsporidia can cause serious disease and are common environmental organisms that have been implicated in waterborne disease outbreaks so attempts have been made to recover microsporidia from water samples for subsequent testing with traditional PCR. Unfortunately, existing molecular procedures are largely uncharacterized, labor intensive, and do not lend themselves to routine use in a water utility laboratory. These experiments addressed these limitations by using an automated nucleic acid extraction method, the MagNAPure LC workstation (MPLC), and a rapid real-time PCR assay, the Encephalitozoon LC assay (LC - PCR) for the LightCycler(TM) instrument (Roche Molecular Biochemicals, Penzburg, Germany). Using laboratory water spiked with spores, a modification of the MagNA Pure LC DNA Isolation Kit I protocol was performed using the automated MPLC. Extracted DNA was amplified using the LC-PCR. Assay detection limits, reproducibility, and efficiency were assessed by comparing threshold cross-over values generated by the LC-PCR. The assay provided rapid reproducible results for detection of microsporidia in laboratory water. The lower limit of detection (LLOD) for spores was less than or equal to 100 spores/ml of laboratory water (i.e., 0.5 spores/ml). PCR efficiency approached 100% for spores in water and was statistically equivalent for spore dilutions ranging from 102 - 107 spores/ml (Tukey-Kramer HSD, Dunnett's test of the means). Assay specificity was 100%, when 39 different enteric organisms were tested. Intra-assay reproducibility was assessed at 104 spores/ml of water and produced a mean C.O. of 33.2 +/- 0.9 with a CV of 2.7 (n=8). Stability of the control spores at 4C was assessed by monitoring the C.O. value as a function of storage time, and proved that DNA concentration for spores in water (106 spores/ml) was relatively stable over a five-month storage period (25.4 +/- 1.4 cycles). By using amplicon melting curve analysis, the assay reproducibly differentiated between E. intestinalis, E. cuniculi, and E. hellem. The Tm for E. hellem, E. cuniculi, and E. intestinalis (alveolar and duodenal strains) were 63.7 +/- 0.2, 67.9 +/- 0.4, 69.7 +/- 0.8, and 69.7 +/- 0.7 C respectively (n=3). Except for E. intestinalis strains, all Tm were significantly different as judged by the 95% CI. No differences were observed in Tm as a function of spore concentration. While there are several published molecular detection assays for microsporidia species in water, this real-time PCR is used to characterize analytical accuracy, reproducibility, and efficiency as well as to enable the differentiation of Encephalitozoon species in a single homogenous real-time PCR system. These methods have the potential to be adapted and safely incorporated into a reference laboratory and provide the water industry with a standardized approach by which other methods can be compared. This method can provide the basis for further testing of different water matrices and concentrated water pellets for the detection of microsporidia in source water. Includes 42 references.