In the pulmonary vasculature, mechanical forces such as cyclic stretch induce changes in vascular signaling, tone and remodeling. Nitric oxide is a potent regulator of soluble guanylate cyclase (sGC), which drives cGMP production, causing vasorelaxation. Pulmonary artery smooth muscle cells (PASMCs) express inducible nitric oxide synthase (iNOS), and while iNOS expression increases during late gestation, little is known about how cyclic stretch impacts this pathway. In this study, PASMC were subjected to cyclic stretch of 20% amplitude and frequency of 1 Hz for 24 h and compared to control cells maintained under static conditions. Cyclic stretch significantly increased cytosolic oxidative stress as compared to static cells (62.9 +/- 5.9% vs. 33.3 +/- 5.7% maximal oxidation), as measured by the intracellular redox sensor roGFP. Cyclic stretch also increased sGCbeta protein expression (2.5 +/- 0.9-fold), sGC activity (1.5 +/- 0.2-fold) and cGMP levels (1.8 +/- 0.2-fold), as well as iNOS mRNA and protein expression (3.0 +/- 0.9 and 2.6 +/- 0.7-fold, respectively) relative to control cells. An antioxidant, recombinant human superoxide dismutase (rhSOD), significantly decreased stretch-induced cytosolic oxidative stress, but did not block stretch-induced sGC activity. Inhibition of iNOS with 1400 W or an iNOS-specific siRNA inhibited stretch-induced sGC activity by 30% and 68% respectively vs. static controls. In conclusion, cyclic stretch increases sGC expression and activity in an iNOS-dependent manner in PASMC from fetal lambs. The mechanism that produces iNOS and sGC upregulation is not yet known, but we speculate these effects represent an early compensatory mechanism to counteract the effects of stretch-induced oxidative stress. A better understanding of the interplay between these two distinct pathways could provide key insights into future avenues to treat infants with pulmonary hypertension.