Purpose To assess the maximum uptake of Iododeoxyuridine (IUdR) by proliferating smooth muscle cells in vitro to determine the optimal concentration to be administrated in an in vivo experiment. The long-term goal is to utilize radioactive IUdR to inhibit smooth muscle cell proliferation and restenosis of arteries after balloon angioplasty in vivo. Methods Porcine smooth muscle cells (SMCs) were cultured in 5% FBS medium and stimulated to proliferate by the addition of medium containing 10% FBS and insulin. IUdR was added at 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, respectively, in proliferating SMCs with control for 1, 3, 5, 7 day incubation. Fluorescence Activated Cell Scanning (FACS) was performed after the SMCs were harvested and double-stained with FITC-conjugated anti-IUdR antibody (B44) and propidium iodide (PI). The ratio of IUdR-labeled cells to total cell population for each IUdR concentration and duration was determined by FACS. All data were repeated three times at each time point. The doubling times, growth curve and cell density of the proliferating SMCs were investigated using Beckman Coulter Particle Counter and digital microscopy. Results The percentage of proliferating SMCs uptaking IUdR increased from 1 to 5 days incubation with all concentrations of IUdR; In day 5, the uptake rate reached the peak value, then decreased by 7 days. IUdR uptake on day 5 was higher with concentrations of 10 μM and 20 μM. The doubling times of the SMCs were prolonged with IUdR concentration increasing, while the proliferating cell number and density compared with control decreased obviously by day 5 (P<0.05).Conclusion The peak time to uptake IUdR was 5 days and optimal concentration of IUdR was between10 μM to 20 μM for proliferating SMCs to uptake in vitro. IUdR itself could inhibit the SMCs' proliferation and the inhibitory effect was related to the concentration.