YC-1 is a direct activator of soluble guanylyl cyclase (sGC) and sensitizes the enzyme for activation by nitric oxide (NO) and CO. Because the potentiating effect of YC-1 on NO-induced cGMP formation in platelets and smooth muscle cells has been shown to be substantially higher than observed with the purified enzyme, the synergism between heme ligands and YC-1 is apparently more pronounced in intact cells than in cell-free systems. Here, we investigated the mechanisms underlying the synergistic activation of sGC by YC-1 and NO in endothelial cells. Stimulation of the cells with YC-1 enhanced cGMP accumulation up to approximately 100-fold. The maximal effect of YC-1 was more pronounced than that of the NO donor DEA/NO (approximately 20-fold increase in cGMP accumulation) and markedly diminished in the presence of L-N(G)-nitroarginine, EGTA, or oxyhemoglobin. Because YC-1 did not activate endothelial NO synthase, the pronounced effect of YC-1 on cGMP accumulation was apparently caused by a synergistic activation of sGC by YC-1 and basal NO. The effect of YC-1 was further enhanced by addition of DEA/NO, resulting in a approximately 160-fold stimulation of cGMP accumulation. Thus, YC-1 increased the NO-induced accumulation of cGMP in intact cells by approximately 8-fold. Addition of endothelial cell homogenate increased the stimulatory effect of YC-1 on NO-activated purified sGC from 1.2- to 3.7-fold. This effect was not observed with heat-denatured homogenates, suggesting that a heat-labile factor present in endothelial cells potentiates the effect of YC-1 on NO-activated sGC.