Circulating levels of platelet activating factor (PAF) are high in the fetus and PAF plays active role in maintaining high pulmonary vascular (PV) resistance in the hypoxic environment in utero (Ibe et al, J Appl Physiol 85: 1998). PAF synthesis by fetal PV smooth muscle cells (SMC) is high in hypoxia, but the effect of oxygen tension on PAF receptor (PAF-r) binding in PV SMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in ovine fetal PVSMC. PAF binding was saturable, with PAF-r density (B_max: fmol/10⁶ cells; means±SEM, n=6) 25.2±0.77 during hypoxia (pO₂ <40 torr), higher than 13.9±0.44 during normoxia (pO₂ ~100 torr). The dissociation constant (KD) in hypoxia was 0.80±0.10, and 2-fold lower than 1.70±0.5 in normoxia. PAF-r protein expression was 35-40% greater in hypoxia, and expression was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting a translational regulation. Inositol phosphate release, used as an index of PAF-r mediated cell signaling, was greater in hypoxia, with EC₅₀ of 2.94±0.61 and 5.85±0.51 nM during hypoxia and normoxia respectively. Furthermore, exogenous PAF induced 50%-90% greater intracellular calcium flux in cells during hypoxia, indicating that hypoxia significantly augments PAF-r mediated cell signaling. Phosphorylation of PAF-r, with or without stimulation with 5 nM PAF, was 40% greater in hypoxia. These data show that in ovine fetal PVSMC, hypoxia significantly up-regulates PAF-r binding, PAF-r phosphorylation as well as PAF-r mediated intracellular signaling, evidenced by augmented inositol phosphate production and intracellular Ca²⁺ flux. Our data show that hypoxia-induced PAF-r phosphorylation results in activation of PAF-r mediated signal transduction. The data also suggest that the hypoxic environment of the fetus facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of a high pulmonary vascular resistance in utero.