Nitric oxide (NO) is an unconventional neuromodulator that signals by intercellular diffusion. Its effects are often mediated by activation of its cytosolic receptor, the hemoprotein soluble guanylyl cyclase (sGC). Regional distribution of heterodimeric (α/β) sGC at both the activity and protein level and its regulation are still unclear. Here, sGC was analyzed in rat brain by Western blot and NO donor-stimulated cyclic GMP accumulation. sGCα₁ and sGCβ₁ immunoreactive protein signals strongly correlated with each other. However, V_max values depended on the type of NO donor used. Sodium nitroprusside, the most widely used compound and formally an NO⁺ donor, was up to 20-fold less effective in stimulating sGC activity than the NO donor diethylamine NONOate. In contrast to the rather even distribution of sGC proteins and SNP-stimulated cGMP accumulation in various regions of rat brain, diethylamine NONOate-stimulated sGC activity varied up to 8-fold between the different brain regions tested. In conclusion, we show that expression of both sGCα₁ and sGCβ₁ subunits is tightly coregulated in rat brain, while yet unknown additional mechanisms affect the V_max of sGC.