External pneumatic compression (EPC) is effective in preventing deep vein thrombosis (DVT) and is thought to alter endothelial thrombo-resistant properties. We investigated the effect of EPC on changes in nitric oxide (NO), a critical mediator in the regulation of vasomotor and platelet function. An in vitro cell culture system was developed to simulate flow and vessel collapse conditions under EPC. Human umbilical vein endothelial cells were cultured and subjected to tube compression (C) (50% reduction in diameter, maximum strain ±10%), pulsatile flow (F) (peak shear of 40 dyn/cm²) or a combination of the two (FC) for 4s intervals once per minute. Control cells were cultured in steady low shear of 0.3 dyn/cm². NO levels were measured with a fluorometric nitrite assay of conditioned media collected hourly. Endothelial nitric oxide synthase (eNOS) mRNA expression was measured by Northern blot at 1 and 6 hours. The data demonstrate that in F and FC groups, there was a rapid release of NO followed by a sustained increase throughout the duration of the experiment. NO production in F and FC were almost identical, while C produced the same low amount of NO as the control group. Conditions F and FC also up-regulate eNOS mRNA expression by a factor of 2.08±0.25 & 2.11±0.21, respectively, at 6 hours. Experiments with different modes of EPC show that NO production responds to the time cycle of compression differently. There was no significant difference in 1 hour NO production. However, during 1 to 6 hours, the production rates with cycle period of 60 sec and 30 sec are much higher than that of 120 sec. The group with 30 sec period has the greatest level of eNOS mRNA expression. These results implicate enhanced NO release as a potentially important factor in the prevention of DVT.