A cultured porcine pulmonary artery model (PA) was used to examine the effects of prolonged NO treatment on the response of this vessel to acutely applied NO and to the alpha adrenoreceptor agonist phenylephrine. 2h treatment with the NO-donor, (Z)-1-(N-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate (DETA-NO) decreased both NO and phenylephrine responsiveness. 24h treatment with DETA-NO resulted in a further reduction in NO responsiveness, but no further reduction in phenylephrine responsiveness. Acute addition of soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) had no effect on phenylephrine responsiveness in PA not treated with DETA-NO. ODQ treatment fully restored phenylephrine responsiveness in PA treated with DETA-NO. sGC₁ subunit protein levels in PA tissue homogenate were 48.6±6.9, 51.6±3.5 and 41.3±2.8 ng/mg total protein for freshly prepared, 2h and 24h NO-treated PA, respectively. Steady state tissue cGMP was not significantly different in the control versus NO-treated PA. sGC specific activity in the absence of added NO was measured in PA homogenate and was 0.29±0.02, 1.38±0.12 and 0.53±0.08 µmol cGMP•min^-1•mg sGC^-1, in freshly prepared, 2h and 24h NO treated PA, respectively. 10 min Hb treatment completely normalized sGC basal activity in homogenates prepared from DETA-NO treated PA and was 0.23±0.02, 0.18±0.03 and 0.25±0.04 µmol cGMP•min^-1•mg sGC^-1, in freshly prepared, 2h and 24h NO treated PA, respectively. The kinetics of the Hb reversal of NO-mediated sGC persistent activation do not support sGC covalent modification as the activation mechanism. We conclude that prolonged NO exposure results in persistent, low level sGC activation and accounts for the resultant persistent alpha adrenoreceptor agonist hyporesponsiveness.