Chromosomal instability in osteosarcoma and its association with centrosome abnormalities. The mechanism that generates the extreme aneuploidy that characterizes osteosarcoma (OS) is poorly understood. In this study, interphase fluorescence in situ hybridization (FISH) analysis was used to enumerate cell-to-cell variation of several different chromosomes. We also investigated whether there was an association between TP53 mutation and centrosome aberrations in the generation of chromosomal aneuploidy in OS in four OS cell lines (HOS, SAOS2, U2OS, and MG63) and in a subset of seven tumors. Our analysis showed that there was a wide range of numerical changes affecting multiple chromosomes in OS cell lines and tumors. These data suggest that chromosomal instability (CIN) could be responsible for the extensive aneuploidy associated with this tumor. The results also showed an increased frequency of atypical mitotic figures in three OS cell lines with defective TP53, function and significantly, a more marked CIN phenotype was present in these lines. Furthermore, numerical aberrations of centrosomes were also present in these three OS cell lines with TP53 mutations. In two of three OS patients' tumors there was a large increase in the percentage of abnormal centrosome numbers. We conclude that CIN is a consistent feature of OS and that an intrinsic disturbance of the chromosomal segregation mechanisms is likely associated with centrosome aberrations.