The intracellular nitric oxide receptor, soluble guanylate cyclase (sGC), is an obligate heterodimer of an αand a smaller, heme-containing β-subunit. The prosthetic heme group is essential for the enzyme's NO-sensing capability and heme-free sGC has been shown to be unesponsive to NO. The generation of a cGMP reporter cell line together with the NO-independent but heme-dependent sGC stimulator BAY 41–2272, the NOand heme-independent sGC activator BAY 58–2667, NO, and ODQ enabled us for the first time to distinguish between both sGC states in intact cells. Investigation of the activation profile of different sGC mutants by transient transfection into this cGMP reporter cell led to the identification of the heme binding motif Tyr135-x-Ser137-x-Arg139 in addition to the axial heme-ligand His105. Mutational analysis of the β hemebinding site identified the amino acids βAsp44, βAsp45 and βPhe74 as being crucially important for the heme-induced sGC activation.