The aim of the present study was to provide a mechanistic insight into how 20-Hydroxy eicosatetraenoic acid (20-HETE) relaxes distal human pulmonary arteries (HPA). This compound is produced by -hydroxylase from free arachidonic acid. Tension measurements, performed on either fresh or 1 day-cultured pulmonary arteries, revealed that the contractile responses to 1 µM 5-hydroxytryptamine (5-HT) were largely relaxed by 20-HETE in a concentration-dependent manner (0.01-10 µM). Iberiotoxin (IbTx) pretreatments (10 nM) partially decreased 20-HETE-induced relaxations. However, 10 µM indomethacin and 3 µM SC-560 pretreatments significantly reduced the relaxations to 20-HETE in these tissues. The relaxing responses induced by the eicosanoid were likely related to reduced Ca²⁺ sensitivity of the myofilaments since free Ca²⁺ concentration ([Ca²⁺ ]) -response curves performed on -escin permeabilized cultured explants were shifted toward higher [Ca²⁺ ]. 20-HETE also abolished the tonic responses induced by phorbol-ester-dybutyrate (PDBu, a PKC-sensitizing agent). Western blot analyses, using two specific primary antibodies against CPI-17 and its PKC-dependent phosphorylated isoform (P-CPI-17), confirmed that 20-HETE interferes with this intracellular process. We also investigated the effect of 20-HETE on activation of Rho-kinase pathway induced-Ca²⁺ sensitivity. Data demonstrated that 20-HETE decreased U-46619-induced Ca²⁺ sensitivity on arteries. Hence, this observation was correlated with an increased staining of p116Rip, a RhoA binding protein. Together these results strongly suggest that the 20-hydroxy-arachidonic acid derivative is a potent modulator of tone in HPA in vitro.