Hypoxic pulmonary vasoconstriction (HPV) is a physiological defence mechanism that, through contraction of pulmonary artery smooth muscle cells (PASMC), enables redistribution of pulmonary blood flow away from areas of lowered O₂ tension toward better-ventilated areas. Increases in [Ca²⁺]_i are primary events in the contraction of PASMC and the current, general concept is that this hypoxia-mediated increase is subsequent to voltage-gated Ca²⁺ entry following inhibition of K⁺ channels (Ward et al. 2004; Archer et al. 2006). Primary cultured, human pulmonary artery smooth muscle cells (hPASMC) were grown in smooth muscle growing medium (Promocell Ltd) under 5% CO₂ at 37^oC. Cells were used within 5 passages and [Ca²⁺]_i imaged using fura-2. Significance (taken as P<0.05) was determined using Student’s t-test. Data shown as mean ± S.E.M. Depolarisation of cells with hypoxia solution (P_O2 ca. 20mmHg) evoked a transient [Ca²⁺]_i elevation that was reduced by 68.8±4.4% by removal of extracellular calcium (+EGTA). Nifedipine (20mM) and verapamil (20mM) attenuated the intracellular [Ca²⁺]_i elevation induced by hypoxia by around 30% suggesting the presence of alternate Ca²⁺ entry pathways. Expression of the Na⁺-Ca²⁺ exchanger, TRPC1 and 6 in hPASMC but not TRPC3, 4, 5 and 7 was found by RT-PCR and confirmed by Western blot. The antagonist of the Na⁺-Ca²⁺ exchanger, KBR9743 (10μM), enhanced, slightly, the [Ca²⁺]_i response to hypoxia, whilst the general antagonists for TRPC, 2APB (100μM) and SKF (40μM), significantly reduced the response to 41.5 ±8.2% and 37.5±3.3% respectively. TRPC6 in hPASMC was knocked out by stealth siRNA (Invitrogen). After 48 hours, the protein levels for TRPC6 were greatly reduced and the hypoxia response was reduced to 21% of the control, suggesting that TRPC6 might mediate the initial response to hypoxia. U73122 (10 µM), an antagonist of PLC-PI and D609 (10 µM), an antagonist of PLC-PK, inhibited 18.2±3.3% and 11.8±3.0% of the [Ca²⁺]_i response to hypoxia, respectively. Considering the proportion of calcium response mediating by TRPC channel, the relatively small effect of PLC suggests other mechanisms must be present to activate TRP channels. AICAR (1mM), the agonist of AMP-activated kinase (AMPK), induced a gradual calcium elevation. Compound C (40μM), the antagonist of AMPK, almost abolished the hypoxia response, suggesting that AMPK may activate TRPC6. Co-immunoprecipitation revealed that AMPK was not co-localised with TRPC6. Taken together, our data support a role for TRPC6 in mediating the [Ca²⁺]_i elevation in response to hypoxia in PASMC and suggest that this response may be linked to cellular energy status via an elevation in AMPK.