Nitric oxide (.NO) is an important intercellular signaling agent throughout the animal kingdom. The majority of the effects of .NO is due to the direct stimulation of soluble guanylate cyclase (sGC). It has been proposed that .NO activates sGC by interacting with a heme moiety on the enzyme. The electron paramagnetic resonance (EPR) spectra of the ferrous-[14N/15N]nitrosyl complexes of the purified enzyme have been obtained. The spectrum of the [14N]nitrosyl-sGC complex was almost identical to that of a 5-coordinate. Hyperfine coupling constants and g-values were determined from computer simulations of the EPR spectra. Electronic absorption spectroscopy was used to show that the 5-coordinate nitrosyl complex on sGC forms under conditions that are typically used to assay activation of the enzyme.