It has been previously reported by us that hypoxia activates lung 15-lipoxygenase (15-LO), which catalyzes arachidonic acid to 15-hydroxyeicosatetraenoic acid (15-HETE), leading to the constriction of pulmonary artery (PA). Rho-associated serine/threonine kinase (ROK), a downstream effector of small GTPase RhoA that may be modulated by G-protein and tyrosine kinase, plays an important role in smooth muscle contraction. However, whether the 15-HETE induced PA vasoconstriction involves the Rho/ROK pathway remains to be demonstrated. Therefore, we studied the contribution of ROK as well as G-protein and tyrosine kinase to the 15-HETE induced pulmonary vasoconstriction using PA ring technique, RNA interference technology, RP-HPLC, western blot and RT-PCR combined with the blockers. The hypoxia-induced expression of ROK is regulated by 15-HETE in rat PA smooth muscle cells (PASMCs), leading to vasoconstriction. The up-regulation of ROK expression caused by 15-HETE appears to be mediated by the G-protein and tyrosine kinase pathways. The translocation of ROK2 from the nucleus to the cytoplasm during hypoxia exposure relies on the mechanism for 15-HETE production. These results suggest that 15-HETE may mediate the up-regulation of ROK expression through G-protein and tyrosine kinase pathways under hypoxic condition, leading to PA vasoconstriction.