Soluble guanylate cyclase (sGC), an intracellular receptor for the ubiquitous biological messenger nitric oxide (NO), is a heterodimer consisting of an alpha- and a heme-containing beta-subunit. Activation of the enzyme upon binding of its physiological activator NO to the heme moiety catalyzes the conversion of GTP to the second messenger cGMP. cGMP regulates various effector systems such as phosphodiesterases, ion channels and protein kinases, thereby modulating many physiological processes including vasodilatation, neurotransmission and platelet aggregation. The pathogenesis of various diseases, especially those of the cardiovascular system, has been linked to inappropriate activation of sGC. The concept of NO-dependent sGC activation as a mechanism underlying antianginal action has been validated by the successful clinical use of NO-releasing drugs for more than a century. Recently, a novel class of NO-independent sGC-stimulators has been identified. These compounds, BAY 41-2272 and BAY 41-8543, stimulate sGC in a NO-independent manner but require the presence of the prosthetic heme moiety. Therefore, these compounds were termed NO-independent but heme-dependent sGC-stimulators. In contrast to these compounds, another very recently identified non-NO-based sGC activator BAY 58-2667 stimulates not only the native sGC but, even more potently, the heme-deficient or oxidized form of the enzyme suggesting a novel mechanism of activation. In the present work the activation mechanism of this novel NO- and heme-independent sGC-activator was investigated. It could be shown that in contrast to NO, BAY 58-2667 does not directly interact with the iron of the prosthetic heme moiety. However, BAY 58-2667 activates the enzyme, like NO, by increasing the maximum catalytic rate. Receptor binding studies indicated that the heme-dependent sGC-stimulators and the heme-independent sGC-activators bind to different independent binding sites at the sGC. For BAY 58-2667 a KD value within nanomolar concentrations was determined. In addition, receptor binding studies suggested the existence of two binding sites for BAY 58-2667 at the sGC. For the first putative binding site an involvement of the tyrosine 371 of the alpha- and the amino acids 231-310 of the beta subunit in the binding of BAY 58-2667 was suggested by a photoaffinity labeling approach. The second putative binding site was localized within the heme binding pocket of sGC. Thereby, the amino acids tyrosine 135 and arginine 139 of the beta subunit of sGC were identified as the anchoring residues of the heme moiety postulated 20 years ago. In addition, a direct competition between BAY 58-2667 and the heme ligand for these two residues could be shown indicating that BAY 58-2667 replaces the native sGC heme moiety. This replacement results in the release of the axial heme ligand histidin 105 and in the subsequent activation of the enzyme. This far-going explanation of the molecular mechanism of the BAY 58-2667-induced enzyme activation will be useful for the synthesis of further NO- and heme-independent sGC-activators. This compounds may replace the organic nitrates nitrates and offer a new approach for treating cardiovascular diseases.