Flowcytometry is a very important technique for the analysis of cell properties, with the advantages of simultaneous multiparameter analysis of large cell population in a short time. Recent advances in computer science and techniques in cell preparation and staining make it more valuable for the study of cell biology and its clinical application. This study was performed to establish the techniques of flowcytometry analysis of osteosarcoma cells, to evaluate the results of the characteristics of the DNA and specific cell cycle phase of osteosarcoma cells obtained by preparation of paraffin-embedded tissue blocks, and to analyze any possible difference between cell populations lacated apart from each other in the tumor mass for making a base for further clinical application. Paraffin-embedded tissue blocks were obtained from 10 cases of primary osteosarcoma, which had undergone amputation without chemotherapy or radiotherapy. Tissue blocks obtained from the most superficial and the deepest portions of the tumor mass from the skin surface were selected respectively in each cases. To evaluate the technique and results obtained, analysis of the whole sample were performed twice in a separate setting. Satisfactory DNA histogram was obtained from 14 of 20 tissue blocks, with the values of distribution in the specific cell cycle phases. DNA aneuploidy was found in 2 cases with a DNA index of 1.6 and 1.3, and no difference in DNA ploidy by the location in tumor mass. The S-phase and G2+M phase fraction were 13.2±8.5 and 6.2±3.1 respectively, reflecting the increased cell proliferation compared with normal cell population. There was no statistically significant difference of these values between superficial and deep portions, but the difference was 9.0±9.7 with a maximum of 26.6, much greater than the difference 3.3±3.6, between the first and second set of analysis. Flowcytometry is a very useful technique in the analysis of the DNA and cell cycle phase properties, and the characteristics of DNA and cell proliferation status of osteosarcoma cells were successfully evaluated by this technique. Unsatisfactory DNA histograms were thought to be the result of inappropriate samples. To adequately evaluate the changes in the tumor mass, standardization in obtaining tumor tissue about the location in the tumor mass is suggested for future studies with flowcytometry about the properties of tumor cells.