ABSTRACT At birth, release of endogenous vasodilators such as nitric oxide and prostacyclin facilitate pulmonary vasodilation via the cyclic nucleotides, cGMP and cAMP. Interaction of cyclic nucleotides and platelet-activating factor (PAF)-mediated responses in pulmonary vascular smooth muscle is not known. We studied the effects of cGMP and cAMP on PAF-mediated responses in ovine fetal intrapulmonary venous smooth muscle cells. Studies were done in hypoxia or normoxia with buffer with 8-Br-cGMP (BGMP) and 8-Br-cAMP (BAMP), as well as cGMPdependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) inhibitors. All groups were treated with 1 nM PAF and incubated for 30 min for the binding assay or 20 min for measurement of inositol 1,4,5-phosphate (IP 3 ) production. BGMP and BAMP decreased PAF binding in normoxia by 63 and 14%, respectively. Incubations with the PKG inhibitor Rp-8-(4-chlorophenylthio)-guanosine-3Ј,5Ј-cyclic monophosphorothioate sodium and the PKA inhibitor Rp-adenosine-3Ј,5Ј-cyclic monophosphorothioate abrogated the inhibitory effects of BGMP and BAMP. PAF-stimulated IP 3 production was 8565 Ϯ 314 dpm/10 6 cells in hypoxia and 5418 Ϯ 118 dpm/10 6 cells in normoxia, a 40% decrease. BGMP attenuated PAFstimulated IP 3 production by 67 and 37% in hypoxia and normoxia, respectively; the value for BAMP was 44% under both conditions. Pretreatment with PKG or PKA inhibitor abrogated BGMP and BAMP inhibition of IP 3 release. PAF receptor (PAFr) protein expression decreased in normoxia, but pretreatment with 10 nM PAF up-regulated PAFr expression. Pretreatment with PAF decreased expression and activities of PKG or PKA proteins in normoxia and hypoxia. Our data demonstrate the existence of cGMP/cAMP-PAF cross-talk in pulmonary vascular smooth muscle cells, which may be one mechanism by which PAFr-mediated vasoconstriction is down-regulated at birth