Background Osteosarcoma is one of the most common primary malignant tumors of bone with poor prognosis.TNF-related apoptosis inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. TRAIL induces apoptosis in various tumor cell lines but is not found to be cytotoxic to many normal cell types in vitro. We investigated the cytotoxic activity of TRAIL and chemotherapeutic agents, including methotrexate (MTX), doxorubicin(DOX) and cisplatin (CDDP), on established osteosarcoma cell line-OS-732.Methods OS-732 cells were incubated with chemotherapeutic agents MTX,DOX and CDDP at various peak plasma concentrations(PPC), 0.1PPC,1PPC and 10PPC, alone or with 100 ng/ml of TRAIL for 24 hours or 48 hours. MTT was used to evaluate the cytotoxic activity of different agents on OS-732. The apoptosis proportion was assayed by flow cytometry. Cellular morphologic changes were observed by phase contrast microscope, scan electron microscope, and transmission electron microscope.Results The inhibitory rate was (24.438±3.414)% with TRAIL of 100 ng/ml for 24 hours. The cells were responsive to DOX and CDDP with a dose-effect relationship (P＜0.05). In OS-732 cells, DOX and CDDP cooperated synergistically with TRAIL when incubated the cells with them for 24 hours (the combined inhibitory rate is (58.360±2.146)% and (54.101 ±2.721)%, respectively). TRAIL alone or drugs alone induced the apoptosis rate was less than 25% (P＜0.05).However, the combination of TRAIL and MTX did not present synergistic effects on OS-732 cells (P＞0.05, compared with TRAIL alone).Conclusions Osteosarcoma OS-732 cells were not responsive to TRAIL-induced apoptosis. DOX and CDDP sensitize osteosarcoma OS-732 cells to TRAIL-induced apoptosis. The combination of TRAIL and MTX presented no synergistic effects on killing OS-732 cells.