The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Since TP receptor occupation with the thromboxane mimetic U46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1-2 mm OD) at basal tension, 5,6-EET (0.3 to 10 µM) induced increases in active tension that were inhibited by Y27632 (1 µM) and HA-1077 (10 µM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle [Ca²⁺]_i was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 µM) induced a contraction that was 7.0 ± 1.6 times greater than without KCl. 5,6-EET (10 µM) also contracted -escin permeabilized PA in which [Ca²⁺]_i was clamped at a concentration resulting in a submaximal contraction. Y27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 µM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 ± 0.03 to 0.30 ± 0.02. Y27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca²⁺ sensitivity of the contractile apparatus.