Poly(epsilon-caprolactone) (PCL) is a biodegradable polyester whose biocompatibility has been widely demonstrated both in vivo and in vitro. In the last few years, our group has confirmed that NaOH-treated PCL films can serve as a suitable biomaterial for vascular tissue engineering by supporting the culture of primary vascular cells and, more recently, endothelial-like EC(2) cells derived from endothelial progenitor cells (EPC). In the present study, NO production in basal conditions and after stimulation with different agents has been evaluated and related to the reactive oxygen species (ROS) content and the intracellular calcium levels on EC(2) cells cultured on NaOH-treated PCL films. The results obtained demonstrate that EC(2) seeded on NaOH-treated PCL films enhance the basal NO levels and show a faster, more intense response to physiological stimuli such as VEGF, bradykinin and thrombin than vein endothelial cells (ECv). This result could be indicative of a better capacity of EC(2) cells to maintain their endothelial functionality when seeded on polymers. On the other hand, the culture of both EC(2) and ECv cells on NaOH-treated PCL films induces a significant increase in both ROS content and intracellular calcium that is balanced out through the stimulation of NO production in these cells. In conclusion, these results demonstrate the ability of NaOH-treated PCL films to support endothelial cell production of nitric oxide and reinforce the idea of considering the endothelial-like EC(2) cells derived from blood progenitors as an adequate source of endothelial cells to functionalize vascular grafts. Furthermore, NaOH-treated PCL films could be considered as a promising cellular NO production-inducing biomaterial for vascular tissue engineering applications.