Tetraploid Meth-A cells were polyploidized by demecolcin, an inhibitor of spindle fibre formation in M phase, and then released from the drug 1, 2, 3 and 4 days after the addition. Octaploid cells were successfully established from cell populations including hexadecaploid cells produced by 2, 3 and 4 days of exposure to demecolcin. One-day-treated cells were polyploidized octaploid cells, but they returned to tetraploid cells. All of the octaploid Meth-A cells showed essentially the same features. The octaploid Meth-A cells had eight homologous chromosomes and double the DNA content of the parent tetraploid cells. The doubling time of octaploid Meth-A cells was 30.2 h, somewhat longer than the 28.3 and 24.0 h of tetraploid and diploid cells, respectively. The fractions of cells in the G1, S and G2/M phases were essentially the same in diploid, tetraploid and octaploid Meth-A cells. The cell volume of octaploid Meth-A cells was about two times that of the tetraploid cells. It was concluded that octaploid Meth-A cells were established from transient hexadecaploid cells produced by the polyploidization of tetraploid cells that had been established from diploid cells.