The heterodimeric heme-protein soluble guanylyl cyclase (sGC) is the only proven receptor for nitric oxide (NO). Recently, two different types of NO-independent soluble guanylyl cyclase stimulators have been discovered. The heme-dependent stimulator 2-[1-[2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5(4-morpholinyl)-4,6-pyrimidinediamine (BAY 41-8543) stimulates the enzyme in a synergistic fashion when combined with NO, requires the presence of the heme group and can be blocked by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)-Oxadiazole-(4,3-a)-quinoxalin-1-one (ODQ). The heme-independent activator 4-[((4-carboxybutyl)[2-[(4-phenethylbenzol) oxy]phenethyl]amino)methyl[benzoic]acid (BAY 58-2667) activates soluble guanylyl cyclase even in the presence of ODQ or rendered heme-deficient. In the present study, BAY 41-8543, BAY 58-2667 and NO strongly increased V(max). Combination of BAY 58-2667 and NO increased V(max) in an additive manner, whereas the synergistic effect of BAY 41-8543 and NO on enzyme activation was reflected in an overadditive increase of V(max). ODQ potentiated V(max) of BAY 58-2667-stimulated soluble guanylyl cyclase. BAY 41-8543 prolonged the half-life of the nitrosyl-heme complex of NO-activated enzyme, an effect that was not observed with BAY 58-2667. These results show the different activation patterns of both compounds and demonstrate their value as tools to investigate the mechanisms that underlie soluble guanylyl cyclase activation.