In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO − ) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO − stimulated PKC-α activity and that subsequently increased p 38 MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p 38 MAPK inhibitor) eliminated ONOO − caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO − induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p 38 MAPK (p 38 MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO − effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO − was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a G i dependent mechanism. This hypothesis was reinforced by G i α phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of G i mediated response) to stimulate adenyl cyclase activity upon ONOO − treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (G i )-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p 38 MAPK axis dependent phosphorylation of its α-subunit (G i α) in the pulmonary artery smooth muscle cells.