DNA-like sequences of the p53 and pRB-inactivating simian virus 40 large T-antigen (SV40 LTag) have recently been found in mesotheliomas in the United States and several European countries. Nuclear expression of SV40 LTag, possibly in concert with detectable telomerase activity, could be responsible for immortalisation of (pre)malignant clones, as suggested by the mesothelioma-specific latency period. Depending on the antibody used, different results have been observed regarding the subcellular expression of SV40 LTag in mesotheliomas with SV40 LTag-like DNA sequences. In this study, we screened 28 Belgian mesothelioma tumour samples for the presence of SV40 LTag-like DNA and its gene product by polymerase chain reaction amplification, using the SV.for3/SV.rev primer set, and by tyramine-amplified immunohistochemistry, using the pAb419 and the pAb101 SV40 LTag antibodies. Amplicons were found in 13 of the 28 (46%) mesotheliomas. Cytoplasmic, but no nuclear, staining was found in 10 of these 13 cases. Although our study confirms the presence of SV40 LTag-like DNA sequences in Belgian mesotheliomas, we did not detect nuclear expression of the viral oncoprotein, which makes a pathogenic role of SV40 LTag in mesothelioma carcinogenesis questionable.