We have previously reported that the sustained phase of hypoxic pulmonary vasoconstriction (HPV) in small intrapulmonary arteries (IPA) of the rat is largely due to Rho kinase-mediated calcium sensitization, and is strongly dependent on extracellular glucose concentration, independent of ATP requirements for contraction. Thus low [glucose] suppressed HPV whilst raised [glucose] (15mM) enhanced it (Leach et al. 2001). We hypothesized that this may relate to preferential support by glycolysis of membrane-delimited activation of Rho kinase. We therefore examined in rat IPA the effects of altered glucose on constriction induced by prostaglandin F2α (PGF2α) (an agonist that acts via Rho kinase), and phosphorylation of MYPT1 (target of Rho kinase) and MLC20. Altering glucose between 0 and 15 mM in normoxia had no effect on constriction elicited by either depolarization with raised [K+] (2-60mM) or PGF2α (1-100 μM) (n=6). However, during hypoxia PGF2α (30μM)-induced constriction was strongly suppressed by reducing glucose from 5 to 2.5mM (72 +/- 8% control, SEM, n=6, p<0.05, Student’s t test) and to 0mM (10 +/- 3%, p<0.01), whereas depolarization-induced constriction was unaffected by 2.5mM glucose, and only reduced to 62 +/- 10% by 0mM (n=6, p<0.05). Consistent with this, removal of glucose during hypoxia reduced MLC and MYPT1 phosphorylation following stimulation with PGF2α to 11 +/- 8% and 8 +/- 3% of that at 5mM glucose (n=11, p<0.001), whereas following depolarization MYPT1 and MLC phosphorylation were only reduced to 47 +/- 11% and 35 +/- 7% (n=12, P<0.01). Altering glucose had no effect on the rise in intracellular calcium induced by PGF2α (n-5). In contrast to HPV, raising glucose to 15mM had no effect on either depolarization or PGF2α induced constriction during hypoxia (n=6). As reactive oxygen species (ROS) are implicated in HPV and we have shown that they activate Rho kinase (Knock et al, 2009), we examined the effects of varying glucose on ROS production using chemiluminescence. ROS production by IPA was reduced in the absence of glucose to 52 +/- 8% of that at 5mM (n=10, P<0.05), but increased by 15mM glucose to 174 +/- 18% (p<0.05). Our results suggest that during hypoxia hypoglycaemia suppresses constriction via inhibition of Rho-kinase mediated calcium sensitization. They also raise the possibility that during HPV at least hyperglycaemia enhances constriction by increasing ROS production.