Malignant mesotheliomas (MMs) are pleural-, pericardial-, or peritoneal-based neoplasms usually associated with asbestos exposure. Mesothelial cells are biphasic and may give rise to epithelial and sarcomatous MMs. In addition, benign or atypical proliferations of mesothelial cells may occur in response to many stimuli. There have been recent reports of simian virus 40 (SV40) DNA large T antigen (Tag) sequences in pleural MMs. To further understand the relationship between SV40, MMs, and mesothelial proliferations, we studied 118 MMs from multiple sites in Germany and North America, including 93 epithelial pleural, 14 sarcomatous or mixed pleural MMs, and 11 peritoneal MMs. In 12 pleural MMs, adjacent noninvasive tumor foci were identified and studied separately. Information about asbestos exposure (detailed history and/or microscopic examination for asbestos bodies) was available from 43 German patients. In addition, 13 examples of reactive mesothelium and 20 lung cancers from the United States were tested. DNA was extracted from frozen tumor and adjacent nontumorous tissues or after microdissection of archival formalin-fixed, paraffin-embedded microslides. Two rounds of PCR were performed with primers SVFor 3 and SVRev, which amplify a 105 bp region specific for SV40 Tag. The specificity of the PCR product was confirmed in some cases by sequencing. Our major findings were: 1) Specific SV40 viral sequences were present in 57% of epithelial invasive MMs, of both pleural and peritoneal origin. No significant geographic differences were found, and frozen and paraffin-embedded tissues were equally suitable for analysis. 2) There was no apparent relationship between the presence of SV40 sequences and asbestos exposure. 3) SV40 sequences were present in the surface (noninvasive) components of epithelial MMs. 4) SV40 sequences were not detected in MMs of sarcomatous or mixed histologies. 5) Viral sequences were present in two of 13 samples (15%) of reactive mesothelium. 6) Lung cancers lacked SV40 sequences, as did non-malignant tissues adjacent to MMs. Our findings demonstrate the presence of SV40 sequences in epithelial MMs of pleural and peritoneal origin and their absence in tumors with a sarcomatous component. Viral sequences may be present in reactive and malignant mesothelial cells, but they are absent in adjacent tissues and lung cancers.