Our previous studies have indicated that transforming growth factor (TGF)-beta1 and VEGF expression are increased in the smooth muscle cell (SMC) layer of the pulmonary vessels of lambs with pulmonary hypertension secondary to increased pulmonary blood flow. Furthermore, we found that TGF-beta1 expression increased before VEGF. Because of the increased blood flow in the shunt lambs, the SMC in the pulmonary vessels are exposed to increased levels of the mechanical force, cyclic stretch. Thus, in this study, using primary cultures of pulmonary arterial SMC isolated from pulmonary arteries of 4-wk-old lambs, we investigated the role of cyclic stretch in the apparent coordinated regulation of TGF-beta1 and VEGF. Our results demonstrated that cyclic stretch induced a significant increase in VEGF expression both at the mRNA and protein levels (P < 0.05). The increased VEGF mRNA was preceded by both an increased expression and secretion of TGF-beta1 and an increase in reactive oxygen species (ROS) generation. In addition, a neutralizing antibody against TGF-beta1 abolished the cyclic stretch-dependent increases in both superoxide generation and VEGF expression. Our data also demonstrated that cyclic stretch activated an NAD(P)H oxidase that was TGF-beta1 dependent and that NAD(P)H oxidase inhibitors abolished the cyclic stretch-dependent increase in VEGF expression. Therefore, our results indicate that cyclic stretch upregulates VEGF expression via the TGF-beta1-dependent activation of NAD(P)H oxidase and increased generation of ROS.