Simian virus 40 (SV40) is a dsDNA polyomavirus that induces osteosarcomas and mesotheliomas in hamsters and transforms many types of cells in tissue culture, including human cells. Osteosarcoma is a bone malignancy with multiple molecular lesions underlining progression from normal bone to osteosarcoma. Recent investigations have identified SV40 DNA sequences in osteosarcomas, suggesting that SV40 may contribute to tumor development. However, these studies also demonstrated that geographical differences exist between SV40 and tumor association. Our study analyzed 46 frozen German tumor specimens (42 osteosarcomas and 4 sarcomas initially suspected to be osteosarcomas) for the presence of SV40 DNA sequences by using PCR. Two different primer sets amplifying a 573 bp region of SV40 Tag gene with the complete intron sequence (SV.for 2/SV.rev) and a 172 bp region with no intron sequence (SV.5/SV.6) were used. DNA sequencing analysis verified the results. No SV40 sequences could be detected using the primer set SV.for 2/SV.rev, while 2 out of 42 osteosarcoma specimens and 1 out of 4 poorly-differentiated tumor specimens contained SV40 sequences, using the primer set SV.5/SV.6. From one of these two positive osteosarcomas, multiple tumor biopsies taken at different times during the dissection, including metastasis, tested positive for SV40. These results indicated that in Germany, only rare osteosarcomas can be linked to SV40. These results support previous findings involving geographical differences in the presence of SV40. Finally, the specific detection of SV40 sequences with multiple specimens from one of the two patients and the absence of SV40 sequences in all other samples underscores the specificity and reproducibility of this investigation and ruled out PCR contamination.