We have studied the effects of partial polyamine depletion, induced by treatment with α-difluoromethylornithine (DFMO) on cell cycle phase distributions in five cultured human carcinoma cell lines. We used flow cytometry of cells stained with chromomycin-A₃ and computer analysis to measure phase distributions of treated and control cultures. All five lines respond to 1–5 mm DFMO treatment with a total absence of measurable putrescine, a loss of >90% of spermidine, and a 30–40% decline in spermine by 48 h after DFMO addition. The proliferation of all five lines is inhibited as well. Nonetheless, only four of the cell lines (HuTu-80, HT-29, MCF-7, and A-427) show a marked increase in the G₁-phase fraction and decrease in the S-phase fraction as a consequence of DFMO treatment. Small, but significant, decreases in the G₂-M populations of these cell lines also occurred after DFMO treatment. Exogenous putrescine (5–50 µm) reversed both the polyamine depletion and the perturbed phase distributions of DFMO-treated cultures but was without effect on phase distributions of cultures not treated with DFMO. The fifth cell line (ME-180) showed no effect of polyamine depletion on cell cycle phase distributions in DFMO-treated cultures and also no effect of exogenous putrescine on phase fractions of either control or DFMO-treated cells. These observations indicate that some human tumor cell lines are dependent upon adequate intracellular polyamine content for maintenance of cell cycle traverse. They also imply that human tumor cell lines are heterogeneous with regard to their cell cycle response to DFMO-induced polyamine deficiency.