PCR-based reverse transcriptase (RT) assays are highly sensitive for broad detection of retroviruses. These assays are currently used for demonstrating the absence of retroviral contamination in vaccines and can also be applied to clinical and laboratory research to investigate low-virus replication. A single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT) has been published that was highly sensitive for retrovirus detection (<10 virions), with enhanced reproducibility and increased efficiency [Sears, J.F., Khan, A.S., 2003. Single-tube fluorescent product-enhanced reverse transcriptase assay with AmpliWax (STF-PERT) for retrovirus quantitation. J. Virol. Meth. 108, 139-142]. In this report, the step-by-step setup and performance of the STF-PERT assay is described and sensitivity, reproducibility and specificity of the assay reported using three different RTs as standards: avian myeloblastosis virus (AMV) RT, murine leukemia virus (MMLV) RT, and human immunodeficiency virus type 1 (HIV-1) RT. Evaluation of virus stocks showed about 1-2 logs difference in RT detection and retrovirus quantitation with the different RT enzyme standards; in general, virus determination using HIV-1 RT was comparable to using the relevant virus RT.