Lactate dehydrogenase-elevating virus (LDEV) induces persistent infections in laboratory mice, alters in vivo physiology, and is a common contaminant of biological materials such as transplantable tumor cell lines. The fluorogenic nuclease reverse transcriptase polymerase chain reaction (fnRT-PCR) assay combines RT-PCR analysis with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. An fnRT-PCR assay specific for LDEV was therefore developed by targeting primer and probe sequences to a unique region of the LDEV nucleocapsid (VP1) gene. Using the LDEV fnRT-PCR assay, we detected only LDEV and did not detect other RNA viruses that are capable of naturally infecting rodents. Using this assay, we detected as little as 10 fg of LDEV RNA; the assay was 10-fold less sensitive when directly compared with the mouse bioassay (measurement of serum LD after inoculation), without the problematic false-positive serum LD enzyme elevations associated with the mouse bioassay. Using the fnRT-PCR assay, we also were able to detect viral RNA in numerous tissues and in feces collected from experimentally inoculated C3H/HeN mice, but we did not detect any viral RNA in similar samples collected from age- and strain-matched mock-infected mice. Finally, using the fnRT-PCR assay, we were able to detect LDEV RNA in biological samples that had previously been determined to be contaminated with LDEV by use of the mouse bioassay and an RT-PCR assay at another laboratory. In conclusion, the LDEV fnRT-PCR assay is a potentially high-throughput diagnostic assay for detection of LDEV in mice and contaminated biological materials.