We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G1/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine- flow cytometric method to more specifically study the G1/S transition, the S phase length, and the progression of cells from S phase through G2+M and into G1, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G1/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G1 phase occurred later than the effect on S phase progression. These results imply that the G2+M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.