In previous studies we demonstrated a novel redox mechanism for directly activating PKG1a and PKAI by H2O2 mediated interprotein disulphide formation. These are examples of how oxidant signalling can directly integrate into phosphoregulation and alter vasotone. We hypothesised that nitric oxide (NO) species may also induce disulphide formation and hence kinase activation through formation of S-nitrosothiol intermediates. The nitric oxide donors SNAP and CysNO were assessed for their ability to generate protein S-nitrosylation in the isolated perfused rat heart using the biotin-switch method. Isolated rat heart perfusion at constant flow with the nitric oxide donor SNAP (100µM-1mM) did not alter protein S-nitrosylation, whereas CysNO (5-20µM) increased this protein modification in a dose-dependent manner. In addition to S-nitrosylation, CysNO also caused a dose-dependent increase in PKG1a and PKA-RI disulphide dimerisation, which was absent in SNAP perfused hearts. However, treatment of heart homogenate in vitro with SNAP induced an increase in both protein S-nitrosylation and PKG1a disulphide dimerisation, indicating that S-transnitrosylation is crucial for increased disulphide formation. When purified recombinant N-terminal PKG1a was exposed to CysNO, it induced intermolecular disulphide formation. This indicates that PKG1a disulphide occurs via formation of an S-nitrosothiol intermediate. It was demonstrated using studies measuring tension in isolated aorta that both NO donors are able to induce dose-dependent vessel relaxation. However, only SNAP mediated relaxation was blocked by the guanylate cyclase inhibitor ODQ, indicating that CysNO induces cGMP independent relaxation. Using PKG and PKA inhibitors in combination with ODQ is was shown that the cGMP independent relaxation generated by CysNO was dependent on PKG and PKA activation. This is consistent with CysNO directly activating PKG and PKA through the formation of a interprotein disulphide bond. These studies highlight the potential for beta-adrenergic like signalling, independently of cAMP elevation, by increased S-nitrosylation of PKA-RI.