Thromboxane A2 (TXA2) is a pulmonary vasoconstrictor that has been implicated in the development of pulmonary hypertension. The present study investigated the involvement of voltage-operated calcium channels (VOCC), the inositol 1,4,5-trisphosphate (IP3) receptor and Rho kinase in the contractile response of rat pulmonary arteries to the TXA2 mimetic, U46619. Concentration response curves were performed in the presence or absence of the VOCC blocker, nifedipine, the IP3 receptor antagonist 2-APB and Rho-kinase inhibition. To investigate the influence of the endothelium on the contractile mechanisms experiments were performed in endothelium-intact and denuded arteries. Male Wistar rats (200-250 g) were killed by cervical dislocation. Ring segments 0.2-0.4cm in diameter were mounted on a small vessel wire myograph with a resting tension of 9.81mN in PSS gassed with 95/5% O2/CO2 at 37°C. Changes in isometric tension were recorded using Powerlab data collection and Chart 5 software. Tissues were allowed to equilibrate for 1 hour then contracted with 60mM KCl then washed. Drugs were incubated for 45mins, and cumulative concentration response curves (CRC) to U46619 constructed. The endothelium was denuded by gentle abrasion of the intimal surface. Results are expressed as a percentage of the KCl-induced contraction and are the means ± S.E.M. Statistical analysis was carried out using Student’s t-test and p < 0.05 is considered significant. The U46619 (1nM-3mM) CRC (pEC50 and Rmax, 7.83 ± 0.04, 130 ± 3%, n=5) was unaffected by nifedipine (1µM) or 2-APB (30µM) in endothelium-intact tissue. Removal of the endothelium shifted the CRC to the left and this curve was shifted to the right and the maximum response reduced by nifedipine or 2-APB and markedly reduced by the combination of nifedipine and 2-APB, U46619 pEC50 and Rmax, -ENDO, 8.11 ± 0.06, 128 ± 4%, n=5; nifedipine, 7.23 ± 0.06, 103 ± 3%, n=5; 2-APB 7.98 ± 0.09, 104 ± 4%, n=5; nifedipine + 2-APB, 6.83 ± 0.12, 57 ± 4%, n=5. The Rho-kinase inhibitor Y-27632 (30µM) caused a rightward shift and a reduced maximum in both endothelium-intact and endothelium-denuded rings. pEC50 and Rmax, +ENDO, 7.83 ± 0.04, 130 ± 3%, n=5; Y-27632, 6.58 ± 0.09, 67 ± 4%, n=5; -ENDO, Y-27632, 7.14 ± 0.05, 89 ± 3%, n=5. The combination of the three in endothelium-denuded rings abolishes the response. This study shows that Rho-kinase is involved in the contractile response to U46619 in endothelium-intact and denuded arteries. In endothelium-denuded but not intact rings the response involves Ca2+ influx through a nifedipine-sensitive VOCC and a 2-APB-sensitive mechanism possibly IP3-mediated Ca2+ release. We have reported previously in intact rings the response to U46619 involves Rho-kinase and a chloride sensitive mechanism.