To examine the alteration in cellular characteristics of polyploid ES cells during long-term culturing, tetraploid H-1 (ES) cells were continuously cultured for 180 days. Cellular DNA content of the tetraploid cells decreased and reached a plateau of 3.3 C, where C represents the complement of haploid chromosomes. The chromosome number also decreased, indicating that the DNA loss was induced by chromosome loss. Cell volume was maintained, suggesting that the DNA loss did not involve cytoplasmic loss. The cell cycle parameters were almost the same during the DNA decay process, indicating that cell cycle progression was independent of the quantity of homologous chromosomes. Hypotetraploid cells showed alkaline phosphatase activity and formed teratocarcinomas in mouse abdomens, suggesting that the pluripotent potential was maintained. Cellular morphology was also retained, suggesting that the gene expression specifying morphological characteristics was conserved. We conclude that these initial cellular characteristics of tetraploid H1 (ES) cells were preserved in long-term culture, irrespective of chromosome loss.