Objective To investigate the clinical value of the test injection at popliteal arteries on 64-slice CTA in lower extremity peripheral arterial occlusive disease (PAOD). Methods Twenty-eight patients with PAOD referred for 64-slice CTA were enrolled consecutively in the study. Test injection was performed at bilateral popliteal arteries (the level of knee joints) and the clinic value of the peak value and the time to peak was analyzed. The relationship between the time to peak and the peak value was evaluated with Pearson test. The time to peak was used for programming of the CT angiographic acquisitions with fast scanning mode. The quality of visualization of each arterial segment was determined independently by two radiologists. Results Fifty-four (96%, 54/56) time-attenuation curves were obtained in 28 patients. The wide interindividual variation in the peak value and the time to peak was observed in 52 curves of 26 patients with range of 60-178 HU,21-46 s and an average of (135±28) HU,(31±6) s, respectively. The difference in the peak value and the time to peak between bilateral popliteal arteries was also observed with range of 10-80 HU and an average of (32±18) HU in 19 patients,with range of 1-12 s and an average of (5±3) s in 21 patients. There was negative relationship between the peak value and the time to peak (r=-0.526, P<0.01). The CTA images were of good (598 segments) or medium quality (12 segments) in 99% segments (610/616). Conclusions The test injection at popliteal arteries was useful for 64-slice CTA in the patients with PAOD, as it could accurately specify the delay time of CT angiographic acquisitions.