Transforming growth factor beta1 (TGFbeta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remain unsettled. We therefore studied TGFbeta1 stimulation of freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGFbeta1 initially (24 h) promoted differentiation, with up-regulated expression of smooth muscle contractile proteins. TGFbeta1 also induced expression of Nox4, the only NAD(P)H oxidase homologue found in HPASMC, through Smad 2/3 signaling but not mitogen-activated protein (MAP)kinases. TGFbeta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGFbeta1, Nox4 was initially present in freshly cultured cells but progressively lost with passage, paralleling a decrease in ROS production over time. At a later time point (72 h), TGFbeta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 siRNA and dominant negative Smad 2/3, indicating that TGFbeta1 produces HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), suggesting that TGFbeta1 may facilitate proliferation by up-regulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homologue in HPASMC. This is the first observation that TGFbeta1 regulates Nox4, with important implications for pulmonary arterial vascular remodeling in vivo.