Despite recent advances, the prognosis of relapsed osteosarcoma patients remains very poor. Application of high energy shock waves may change the tumour cell growth and increase the cytotoxic effect of in vivo and in vitro chemotherapeutic agents. We studied the effect of their association with doxorubicin or methotrexate on three in vitro osteosarcoma cell lines. The effect of these combinations on SJSA-1, MG-63 and HOS human osteosarcoma cell lines were evaluated through incubation with doxorubicin or methotrexate and high energy shock wave treatment with 1000 shots at 0.22 mJ/mm(2) and an evaluation of the cell number, cell proliferation and apoptosis at days 1, 3 and 6 from the start of treatment. The combination of high energy shock waves and doxorubicin induced a statistically significant advantage in terms of a decrease in cell number on the SJSA-1 and HOS cell lines, a decrease of cell proliferation on all three cell lines and an increase of apoptosis only on the SJSA-1 cell line. The combination of high energy shock waves with methotrexate induced a decrease of the cell number only in the SJSA-1 and in the HOS cell lines, of the cell proliferation in the SJSA-1 and in the MG-63 cell lines, and an increase of apoptosis only on the SJSA-1 cell line. In conclusion, these experiments show an interesting effect of high energy shock waves on osteosarcoma cell lines, which could lead to future studies of the in vivo effects of high energy shock waves in the murine model as well.