Chronic exposure to hypoxia such as at high altitude causes pulmonary hypertension (PH). PH is characterised by remodelling of pulmonary vessels and sustained increases in pulmonary vascular resistance (PVR)(1, 2). Vasoconstriction was previously viewed as playing a minor role in chronic PH, with remodelling of the pulmonary vessels accounting for the majority of the elevation in PVR. More recently studies have shown that in the rat increased PVR in the chronically hypoxic lung is almost entirely due to rho kinase dependent vasoconstriction (3). Knockout and transgenic mice are increasingly commonly used to investigate of PH. Therefore we developed an approach combining the isolated perfused lung technique and stereology to determine the contributions of vasoconstriction and remodelling to PVR in the hypoxic mouse. Wild type mice were exposed to hypoxia (10% O2) or normoxia (21% O2) for three weeks following which they were deeply anaesthetized (sodium pentobarbital 60mg.kg-1, i.p.) and exsanguinated. The lungs were removed and resistance assessed using an isolated ventilated perfused preparation (n=17-20 per group). In a subset of lungs (n=8 per group), the rho kinase inhibitor Y27632 (10-5M) was added to the perfusate and resistance assessed pre- and post-vasodilator addition. The increase in PVR in the hypoxic group was expressed as a fraction of the mean normoxic resistance. Separate groups of lungs were isolated and fixed at standard airway and vascular pressures for quantitative stereological assessment of vascular structure as previously described(4). The reduction in lumen diameter in each hypoxic lung was expressed as a fraction of the mean normoxic value and the resultant change in PVR calculated using Poiseuille’s equation. In chronic hypoxic mice the mean increment in PVR was 0.85(±0.032)* of the mean normoxic value in control lungs. The rho-kinase inhibitor Y27632 significantly (P<0.01) reduced PVR by 0.37(±0.04)*, but did not restore it to normoxic levels. The remaining increment above the mean normoxic value was taken to be due to structural changes (0.47±0.04)*. Total intra-acinar vessel length was unchanged following chronic hypoxia and therefore the structurally determined increase in PVR was modeled as solely due to a reduction in lumen diameter. This calculated structural component (Poiseuille’s equation) was 0.54(±0.089), which was not significantly different from the hemodynamically determined value. In conclusion, two mechanisms contribute approximately equally to the elevation in PVR in the chronically hypoxic mouse lungs. Firstly, rho-kinase mediated vasoconstriction and secondly, structural lumen narrowing of the pulmonary vessels. * significantly different from 0 (P<0.01)