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The effects of culture conditions on the distribution of chromosome number in mouse Sarcoma 180 Y.S. cells were studied in respect to heterogeneity of their chromosome number and their tumorigenicity; four subpopulations separated by ficoll gradient centrifugation from in vivo cultured tumor cells demonstrated different tumorigenic properties and the hypotetraploid cells were found to be the most tumorigenic. The results summarized are as follows 1. The tumor cell populations were heterogeneous in their chromosome number and their tumorigenicity, and these heterogeneity could be changed by different culture conditions. The chromosome numbers of tumor cells cultured in vivo were varied from 15 to 140 with modal number of 110-120, while those cultured in vitro ranged from 60 to over 300 with modal number of 121-130. Frequency distribution of hypotetraploid cells in in vivo cultures was 22.3% whereas it was 2.4% in in vitro cultures. 2. When the tumor cells were cultured in vitro and inoculated into the peritoneum of mice, the frequency of cells with high chromosome number of more than 300 (15n) was increased from 1.2% to 8.6% and 33.1% as the passage number increased. Hypotetraploid cells were increased along with serial in vitro to in vivo passage. 3. Tumor producing ability in cells cultured in vitro was increased from 20% to 25% and 40% in accordance with serial in vivo passage thereafter. It is, therefore, suggested that altered characteristics of tumor cells cultured in vitro appeared to be reversed into their original properties by maintaining them in vivo. 4. When the tumor cells were separated by 10%, 15% and 20% ficoll concentration gradient, the highest relative frequency of hypotetraploid cells were found in 10% ficoll concentration. Fifty percent survival days of mice inoculated with these subpopulations were 25.3 days, 28.7 days and 29.4 days for each subpopulation separated by the concentration of 10%, 15% and 20% ficoll respectively. From the above results it is suggested that cells with high hypotetraploid among the heterogeneous tumor cell population appeared to be an important factor responsible for tumor producing ability.