The pulmonary circulation responds to acute moderate hypoxia with an immediate vasoconstriction (HPV), which helps to match pulmonary ventilation and perfusion and optimises gas exchange in the lung. In this study we have investigated the potential role of mitochondrial depolarisation in HPV using FCCP. Small intrapulmonary arteries (IPA) from male Wistar rats (200-300g) were dissected free and mounted on a Mulvany-Halpern myograph, bathed in Krebs solution and gassed with 5% CO₂, balance air. In order to evoke HPV vessels were preconstricted with PGF_2α and subsequently exposed to 5% CO₂, balance nitrogen. This evoked a biphasic contraction of the pulmonary arteries with a rapidly developing transient and a slow sustained (phase 2) contraction. Phase 2 HPV was nearly abolished by preincubation with the ryanodine receptor blockers dantrolene (50 μM; 62% inhibition, P<0.001, n=10) and ryanodine (100 μM; complete inhibition, P<0.001, n=3) and the SOCC inhibitor 2-APB (75 μM; 60% inhibition, P<0.001, n=5), indicating an important contribution of intracellular Ca²⁺ release via ryanodine receptors and Ca²⁺ influx via a non-voltage-dependent pathway. FCCP evoked a biphasic contractile response in PGF_2α–preconstricted pulmonary arteries, which strongly resembled HPV in size and time course. In mesenteric resistance arteries FCCP produced a transient, but not a sustained contraction (n=8). In analogy to HPV, the FCCP–induced contraction in IPA was attenuated by preincubation with dantrolene (74% inhibition, P=0.03, n=3) or 2-APB (complete inhibtion, p=0.02, n=4). Moreover, preincubation of IPA with FCCP strongly attenuated the development of the sustained contraction during hypoxia (58% inhibition, P<0.01, n=5). However, the FCCP response differed from that to hypoxia in that it was strongly inhibited by 10 μM diltiazem (64% inhibition, P=0.04, n=4) and also by 30 μM cyclopiazonic acid (73% inhibition, P=0.01, n=5), neither of which inhibited sustained HPV. Therefore, although FCCP inhibited phase 2 HPV, and caused a contraction in IPA which in some ways mimicked this response, the results suggest that mitochondrial depolarisation is unlikely to be a major factor in causing HPV. The results indicate also that mitochondrial Ca²⁺ content in IPA is strongly influenced by plasmalemmel and sarcoplasmic reticulum Ca²⁺ homeostasis.