Cultivated arterial smooth muscle cells were used to study the effects of platelet-derived growth factor (PDGF) on polyamine synthesis and of polyamine synthesis inhibitors on initiation of DNA synthesis by PDGF. Quiescent (serum-starved) cells showed no detectable activity of ornitihine decarboxylase (ODC), the first and overall rate-limiting enzyme in polyamine synthesis, and had low levels of the polyamines putrescine, spermidine and spermine. PDGF caused a marked increase in ODC activity, with a peak after 6 h followed by a rapid decline. The rise in enzyme activity was blocked by actinomycin D and cycloheximide, suggesting control at the transcriptional and translational levels, α-Difluoromethylornithine (DFMO), a catalytic and irreversible inhibitor of ODC, prevented the appearance of enzyme activity. The cellular content of putrescine increased distinctly in response to PDGF, with a peak after 8 h, abolished by simultaneous treatment with DFMO. In contrast, the concentrations of spermidine and spermine changed but little within the 20-h period examined here. DFMO inhibited the initiation of DNA synthesis without affecting the length of the prereplicative lag phase and was fully active if added within 4 h after PDGF. The effect of DFMO on DNA synthesis was counteracted by addition of polyamines to the culture medium and exogenous polyamines were also found to stimulate DNA synthesis in PDGF-free medium (spermine > spermidine > putrescine). It is concluded that induction of ODC and putrescine synthesis are integral parts in the mitogenic response of arterial smooth muscle cells to PDGF.