Osteosarcoma is a primary bone malignancy that typically occurs during adolescence but also has a second incidence peak in the elderly. The etiology of osteosarcoma is not well understood. Recent investigations have identified SV40 DNA sequences in osteosarcomas, suggesting that SV40 may contribute to tumor development. However, these studies also demonstrated geographical differences in SV40-positive osteosarcomas. The purpose of this study was to determine the prevalence and clinicopathological characteristics of SV40 positive osteosarcoma in Tunisian patients. Fifty-six formalin-fixed paraffin-embedded specimens of osteosarcomas were retrospectively investigated. Samples investigated were clinical cases examined between 1990 and 2004 in the Laboratory of Pathology at the University Hospital Farhat-Hached of Sousse (Tunisia). The search for SV40 was performed by immunohistochemistry using the Pab108 antibody for the detection of the viral oncoproteins: large T antigen and small t antigen (T/t-ag). SV40 status was correlated with clinico-pathological data. T/t-ag immunostaning was detected in the tumor cells in 31/56 (55.4%) osteosarcoma cases. SV40 positivity was more frequent (83%) in patients older than 40 years (5/6 cases) than in patients under 40 years (52%, 26/50), but the difference does not reach statistical significance (p = 0.33). Moreover, the time between the onset of clinical symptoms and diagnosis was shorter for SV40 positive than SV40 negative cases (p = 0.08). However, the viral status did not differ significantly according to gender, tumor size, histological subtype, tumor location, or metastases. This study documents the presence SV40 T/t-antigens in a proportion of osteosarcomas in Tunisian patients. The expression of these viral oncoproteins supports the hypothesis that SV40 may have a role in the pathogenesis of this tumor.