Whereas the systemic circulation dilates in response to hypoxia, the pulmonary responds with a biphasic constriction. The latter is termed hypoxic pulmonary vasoconstriction (HPV; von Euler & Liljestrand, 1946). Physiologically, HPV maintains the ventilation-perfusion ratio during alveolar hypoxia. Pathologically, ongoing HPV contributes to pulmonary hypertension. Most groups require preconstricting agonists, such as PGF2α, to elicit HPV in isolated arteries. However, the mechanisms underlying the agonist-induced contraction may overlap with those of HPV, so we have characterised HPV in unpreconstricted arteries. Intrapulmonary arteries (500-800 μm dia.) from male Wistar rats were mounted on a myograph. Drugs were applied 20 min before hypoxia (95% N2/5% CO2; pO2=15-20 Torr). All data were analysed using repeated measures 2-way ANOVA with Holm-Sidak’s post-hoc test. The L-type Ca2+ antagonists diltiazem (10 μM; n=5) and nifedipine (3 μM; n=5), and the store-operated Ca2+ channel antagonist La3+ (100 μM; n=4) had no significant effect on HPV. Furthermore, HPV persisted in Ca2+-free Krebs containing EGTA (200 μM; n=4; n.s.). Both ryanodine (100 μM; n=4; P < 0.05) and the lysosomal H+-ATPase inhibitor concanamycin A (1 μM; n=5; P < 0.05) strongly suppressed HPV. Although it has been proposed that cyclic ADP-ribose (cADPR) and AMP-activated protein kinase (AMPK) mediate hypoxia-induced Ca2+ release from the SR (Evans et al., 2005), neither the cADPR antagonist 8-bromo-cADPR (30 μM; n=4) nor the AMPK inhibitor compound C (10 μM; n=4) had a significant effect on HPV. The rho-kinase inhibitor Y-27632 (1 μM) markedly attenuated HPV (n=5; P < 0.05) and the hypoxia-induced biphasic (at 2 and 30 min) increase in phosphorylation of the myosin phosphatase targeting subunit MYPT-1 (n=6; P < 0.05) and the monophasic increase (at 30 min) in phosphorylation of the 20 kDa regulatory myosin light chain (MLC20; n=6; P < 0.05). Superoxide dismutase (SOD; 200 units ml-1; n=6; P < 0.05) but not catalase (200 units ml-1; n=5; n.s.) suppressed HPV. Removal of the endothelium strongly inhibited HPV (n=5; P < 0.05). These data are consistent with a model in which HPV in these arteries is dependent upon the endothelium, but does not require cADPR, AMPK or extracellular Ca2+ influx. Conversely, HPV appears to involve superoxide, Ca2+-sensitisation via rho-kinase and intracellular Ca2+ release from concanamycin and ryanodine sensitive compartments.