We evaluated changes in DNA synthesis, structure, and mechanical activity in isolated arteries during exposure to growth factors. Renal arteries were isolated from rats, sympathectomized, denuded of endothelium, and maintained in tissue culture. Up to 4 days of culture did not affect maximal contractile responses to depolarization. From the results of nuclear incorporation of the thymidine analogue 5-bromo-2-deoxyuridine (BrdUrd), culture stimulated DNA synthesis. In the media, incorporation of BrdUrd was maximal after 3 days but fell precipitously thereafter. Culture of arterial segments did not, however, increase the cross-sectional area of the media, the ploidy of the arterial nuclei, or the number of medial cells. In contrast, new layers of cells, part of which displayed smooth musclelike properties, developed at the border of the segments. The outermost edge of this newly formed layer continued to incorporate BrdUrd for at least 2 wk. These data demonstrate that stimulation of DNA synthesis by continuous exposure of the arterial wall to exogenous growth factors is 1) transient in the media; 2) does not, at least initially, compromise contractile reactivity; 3) does not alter gross medial structure; but 4) leads to proliferation of smooth musclelike cells outside the media. These findings suggest that the number of smooth muscle cells in the arterial media is maintained constant in the presence of even strong mitogenic stimuli.