The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to NO-induced NO hyporesponsiveness in the pulmonary artery. A cultured porcine pulmonary artery model (PA) was developed to examine the effects of prolonged NO treatment on the response of this vessel to acutely applied NO (NO responsiveness). The DNA, total RNA, expression of actin and myosin and maximum isometric force induced by the alpha adrenoreceptor agonist phenylephrine were unchanged following up to 48 h in culture medium. 24 h treatment with the NO-donor, (Z)-1-(N-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate (DETA-NO) resulted in no significant change in these characteristics from that of both freshly obtained and 24 h cultured PA. Following 24 h exposure to DETA-NO, PA contracted to approximately 50% of maximal isometric force with phenylephrine was less responsive to acutely applied DETA-NO (-log EC₅₀ 6.55 ± 0.12 and 5.02 ± 0.21, respectively). The reduction in NO-responsiveness with 24 h DETA-NO treatment depended upon the concentration of the NO-donor used and was not observed in PA treated for 24 h with 1 µM DETA-NO. Treatment of PA with the cell permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (MnTBAP) resulted in no significant change in either the response to phenylephrine or in DETA-NO EC₅₀ in both freshly prepared and 24 h DETA-NO treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA, 247 ±15 and 235 ± 42 pmol·min^-1·mg^-1, respectively. 24 h DETA-NO treatment resulted in no significant change in either cGMP or cAMP phosphodiesterase activities. mRNA expression for the two soluble guanylyl cyclase (sGC) subunits found in porcine PA, sGC₁ and sGC₁, as determined by quantitative RT-PCR was 0.29 ± 0.08 and 0.19 ± 0.03 attomoles/µg total RNA, respectively. 24 h in culture had no significant effect on sGC subunit mRNA expression, but 24 h DETA-NO treatment resulted in a significant reduction in the mRNA expression of both sGC subunits (0.16 ± 0.03 and 0.10 ± 0.02 attomoles/µg total RNA for sGC₁ and sGC₁, respectively). sGC protein expression, as determined using semiquantitative immunoblotting, was 42 ± 4 ng/mg soluble protein. 24 h in culture resulted in a small reduction and 24 h DETA-NO treatment resulted in a further reduction in sGC protein expression (36 ± 3 and 31 ± 3 ng/mg soluble protein, respectively, p < 0.025). Basal tissue cGMP (cGMP)_i was significantly increased and NO-induced (cGMP)i was significantly decreased by 24 h DETA-NO treatment. When (cGMP)i was normalized to the amount of sGC protein expressed in PA, it was significantly lower in PA treated for 24 h with DETA-NO compared to both freshly isolated and 24 h cultured PA (3.69 ± 0.45, 5.24 ± 0.81 and 6.95 ± 0.70 pmol cGMP· 10 min^-1·ng sGC₁^-1, respectively, P < 0.05). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing sGC mRNA and protein expression and by decreasing sGC specific activity.