Although polyamine (PA) levels are believed to increase in response to mitogenic stimuli in all cells during growth, their role in arterial smooth muscle cell (ASMC) proliferation, an essential step in atherogenesis, is unknown. To determine whether the arterial wall mitogen, platelet-derived growth factor (PDGF) influences PA metabolism when cell cycle traverse is initiated, we examined its effects on the transport of the PA precursor [3H]putrescine (PUT) in culture in bovine and human ASMC. PUT uptake was stimulated in a dose-response relationship by PDGF-containing human serum (2-10%), and abolished in 24 h without it. Inhibition of this uptake by spermidine and the lack of effect of thymidine, leucine and ornithine indicated that uptake was by a PA-specific mechanism. Without serum, platelet releasate containing PDGF stimulated PUT uptake but not that of leucine or glucose. While platelet-poor plasma alone also promoted PUT uptake, the combination of platelet releasate and platelet-poor plasma was required for maximal DNA synthesis. PUT uptake under these conditions reached a peak at 16 h, while the synthesis of DNA was maximal at 24 h. Supraphysiological concentrations of insulin, and fibroblast and epidermal growth factors, also stimulated the uptake of the labeled PUT both in the absence and presence of serum, but at much lower rates than those observed with platelet releasate. These findings indicate that the early replicative actions of a variety of mitogens for ASMC involve stimulation of PUT uptake and suggest that PA uptake must precede the initiation of DNA synthesis in ASMC during atherogenesis.