Nitric oxide (NO) is a physiologically relevant activator of the hemoprotein soluble guanylate cyclase (sGC). In the presence of NO, sGC is activated several hundred-fold above the basal level by a mechanism that remains to be elucidated. The heme ligand n-butyl isocyanide (BIC) was used to probe the mechanism of NO activation of sGC. Electronic absorption spectroscopy was used to show that BIC binds to the sGC heme, forming a 6-coordinate complex with an absorbance maximum at 429 nm. BIC activates sGC 2–5-fold, and synergizes with the allosteric activator YC-1, to activate the enzyme 10–20 fold. YC-1 activates the sGC-BIC complex, and leads to an increase in both the Vmax and KM. BIC was also used to probe the mechanism of NO activation. The activity of the sGC-BIC complex increases 15-fold in the presence of NO, without displacing BIC at the heme, which is consistent with previous reports that proposed the involvement of a non-heme NO binding site in the activation process.