Oxidative stress is associated with a number of cardiovascular diseases including pulmonary hypertension (Liu et al., 2006). Previous studies have shown that prostaglandin-F2α and hypoxia both activate the Src family of non-receptor tyrosine kinases (SrcFK) and enhance SrcFK-dependent total cellular tyrosine phosphorylation and contraction, via activation of Rho-kinase (Knock et al., 2008a, Knock et al., 2008b). We further hypothesise that SrcFK mediate these responses through interaction with reactive oxygen species (ROS) production.Isometric tension and protein phosphorylation responses were recorded in freshly isolated intra-pulmonary arteries (IPA) of male Wistar rats. Intra-cellular translocation of RhoA and measurement of ROS production were measured in primary cultures of rat pulmonary arterial smooth muscle cells (PASMC). ROS was measured using the chemi-luminescent probe L-O12. RhoA translocation was visualised using PASMC stably transfected with GFP-tagged RhoA. Data are expressed as mean ± SEM, and compared by ANOVA.SrcFK auto-phosphorylation (at tyr-416) was enhanced by exogenous H2O2 (10µM) in a time-dependent manner (2min: 152 ± 21% of control, n=10, p<0.05 vs. control; 5min: 161 ± 22% of control, n=8, p<0.01 vs. control) and by the superoxide producing quinone LY83583 (1µM) in a time-dependent manner (30sec: 162 ± 15% of control, n=8, p<0.01 vs. control; 1 min: 142 ± 15% of control, n=8, p<0.05 vs. control; 5min: 144 ± 23% of control, n=8, p<0.05 vs. control).The thromboxane analogue U-46619 (100nM, 30min) also enhanced SrcFK auto-phosphorylation (155 ± 14% of control, n=10, p<0.01 vs. control), and enhanced phosphorylation of the Rho-kinase substrate myosin phosphatase targeting subunit-1 (MYPT-1, at thr-855; 143 ± 9% of control, n=16, p<0.01 vs. control) and myosin light-chain-20 (MLC20, at ser-19; 189 ± 21% of control, n=20, p<0.01 vs. control). Pre-incubation with the antioxidant tempol (3mM, 10min) significantly inhibited U46619-induced Src phosphorylation (95 ± 13% of control, n=6, p<0.01 vs. U46619 alone) U46619-induced MYPT-1 phosphorylation (120 ± 5% of control, n=8, p<0.05, vs. U46619 alone) and U46619-induced MLC20 phosphorylation (117 ±15% of control, n=10, p<0.05, vs. U46619 alone). Tempol had no effect on basal phosphorylation of any of the three proteins (Src, n=7; MYPT-1, n=7; MLC20, n=10). The SrcFK antagonist PP2 (30µM, 10min) also inhibited U46619-induced SrcFK phosphorylation (61 ± 4% of control, n=5, p<0.01 vs. U46619 alone), U46619-induced MYPT-1 phosphorylation (103 ± 8% of control, n= 13, p<0.01 vs. U46619 alone) and U46619-induced MLC20 phosphorylation (112 ± 11% of control, n=15, p<0.01 vs. U46619 alone). PP2 significantly inhibited basal phosphorylation of SrcFK (51 ± 19% of control, n=4, p<0.05 vs. control), but had no effect on basal MYPT-1 or MLC20 phosphorylation.U46619 induced contractile responses in IPA and these responses were significantly inhibited by the antioxidants tempol (3mM) (25 ± 5% relaxation, n=11, p<0.01 vs. DMSO control) and ebselen (10µM) (30 ± 4% relaxation, n=12, p<0.01 vs. DMSO control). U46619 (100nM, 30min) induced sustained increases in ROS production in PASMC (216 ± 51% of control, n=8, p<0.01 vs. control) and this was inhibited by 3mM tempol (22 ± 6% of control, n=7, p<0.01 vs.U46619 alone) and 10µM ebselen (79 ± 33% of control, n=5, p<0.01 vs. U46619 alone). U46619 triggered reversible cytosol to cell periphery translocation of GFP-tagged RhoA in live PASMC (n=>25 cells). This translocation was prevented by pre-incubation with 1µM ebselen (n=3), 3mM tempol (n=4) and 10µM PP2 (n=3).Our data show that: U46619 induces ROS production in PASMC; that exogenous ROS activate SrcFK; and that U46619-induced SrcFK, RhoA and Rho-kinase activity and contraction in IPA and PASMC are all ROS and Src-dependent. Src may therefore be acting as a ROS-sensitive intermediary for the activation of the RhoA/Rho-kinase Ca2+-sensitization pathway in response to vasoconstrictor stimuli in rat pulmonary artery.