Soluble rat liver guanylate cyclase purified with GTP-Mn2 ion affinity chromatography has a pI of 6.1 to 6.3 in analytical polyacrylamide gel isoelectrofocusing. The purified enzyme was activated by nitrohemoglobin as well as by an endogenous activator, and maximally activated in the presence of both together. Work on purification of this activator and of a low molecular weight heat-stable factor that permits nitroprusside activation of the guanylate cyclase is in progress.