In the vascular system, the receptor for the signaling molecule NO, guanylyl cyclase (GC), mediates smooth muscle relaxation and inhibition of platelet aggregation by increasing intracellular cyclic GMP (cGMP) concentration. The heterodimeric GC exists in 2 isoforms (α₁-GC, α₂-GC) with indistinguishable regulatory properties. Here, we used mice deficient in either α₁- or α₂-GC to dissect their biological functions. In platelets, α₁-GC, the only isoform present, was responsible for NO-induced inhibition of aggregation. In aortic tissue, α₁-GC, as the major isoform (94%), mediated vasodilation. Unexpectedly, α₂-GC, representing only 6% of the total GC content in WT, also completely relaxed α₁-deficient vessels albeit higher NO concentrations were needed. The functional impact of the low cGMP levels produced by α₂-GC in vivo was underlined by pronounced blood pressure increases upon NO synthase inhibition. As a fractional amount of GC was sufficient to mediate vasorelaxation at higher NO concentrations, we conclude that the majority of NO-sensitive GC is not required for cGMP-forming activity but as NO receptor reserve to increase sensitivity toward the labile messenger NO in vivo.