SV40 viral DNA sequences, particularly large T-antigen (T-ag) DNA, have been reported in malignant pleural mesothelioma (MPM) and suggested to play a role in the tumorigenesis of this cancer. These results remain somewhat controversial owing to variability among a number of laboratories in reported SV40 DNA and protein detection in MPM tissues. This could be explained in part if SV40 DNA is present in relatively low abundance in many MPM tissues determined to contain viral sequences and is therefore difficult to detect. To this end, we investigated the efficacy of real time quantitative PCR in detecting low copy number SV40 DNA sequences, then we quantified SV40 copy number in MPM tissues and cell lines at our institution. Quantitative PCR demonstrated consistent precision and accuracy in detecting SV40 DNA sequences over a large logarithmic range of viral copy number. In addition, SV40 sequences were found in 2/35 tissues and 3/7 cell lines in relatively low abundance. We conclude that SV40 is not a contributing factor in the pathobiology of the majority of MPM tumors from patients at our institution.