In this paper we demonstrate that compounds that promote stomatal opening such as kinetin, atrial natriuretic peptide (ANP) and plant natriuretic peptide immunoanalogues (irPNP) significantly elevate cGMP in guard cell protoplasts. Stomata opened by irPNP are induced to close in the presence of the guanylate cyclase inhibitor, LY 83583. The effect of cGMP on stomatal opening appears to be linked with Ca2+ levels. ANP, irPNP and 8-Br-cGMP all induce stomatal opening and this is inhibited by compounds that lower intracellular Ca2+ levels such as ethylene glycol bis(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), ruthenium red and procaine. This connection between Ca2+ and cGMP is further supported by the fact that irPNP induced increases in cGMP levels do not occur in the presence of EGTA. Since the plasma membrane H+-ATPase is a key enzyme driving stomatal opening, we determined if a causal relationship exists between cGMP, ANP or irPNP and proton transport across the guard cell. Our results showed that the activity of the H+-ATPase is reduced by 8-Br-cGMP and increased by ANP and irPNP. However, ATP-dependent transmembrane H+ gradients are only increased with ANP and not irPNP. This irPNP response can be explained by a direct or indirect irPNP-dependent activation of the enzyme that does not translate into an increase in proton gradient, possibly because irPNP affects H+ coupled symporters.