We determined the clinical value of flow-cytometric measurement of tumor-cell DNA content, which reflects the chromosome number (ploidy), in patients with osteosarcoma of an extremity. Hyperdiploid stem lines were identified in 25 of 26 tumor samples obtained at diagnosis from patients who did not have clinically overt metastases. Near-diploid tumor stem lines coexisted with hyperdiploid lines in 15 of these 25 cases; an isolated near-diploid line was present in the 26th case. All 26 patients underwent definitive surgery and then were treated uniformly with intensive adjuvant combination chemotherapy. Kaplan-Meier analysis of both relapse-free and overall survival times showed that the presence of a near-diploid tumor stem line was associated with improved outcome (P = 0.003 for each comparison). After a median follow-up time of three years, pulmonary metastases developed in only 2 patients in the group with near-diploid lines, in contrast to 7 of the 10 with hyperdiploid lines exclusively. Near diploidy remained significantly associated with improved relapse-free survival after adjustment for the influence of age, the only clinical variable that showed prognostic strength in this analysis (P less than 0.01; relative risk, 0.08; 95 percent confidence interval, 0.02 to 0.48). Our findings demonstrate the usefulness of flow-cytometric determination of tumor-cell ploidy for predicting the sensitivity of histologically high-grade osteosarcoma to chemotherapeutic agents. Patients with a near-diploid tumor stem line can be expected to respond favorably to adjuvant chemotherapy as used in this study, whereas those with only hyperdiploid lines should be considered as candidates for alternative therapy.