We investigated whether a complete inhibition of nitric oxide (NO) formation caused by bacterial endotoxin (lipopolysaccharide, LPS) in vivo prevents the hypotension and restores the vascular hyporeactivity to normal in vivo and ex vivo. The combination of dexamethasone (Dex; 3 mg/kg at 30 min before LPS) plus aminoguanidine (AG; 15 mg/kg at 2 h after LPS) inhibited the overproduction of nitrate (an indicator of NO) in the plasma and aortic smooth muscle and also prevented the development of the delayed hypotension in rats treated with LPS for 6 h. However, the vascular hyporeactivity to norepinephrine (NE) was only partially improved either in vivo or ex vivo in endotoxemic rats treated with Dex plus AG. Pretreatment of aortic rings with N-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxidazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced the contraction to NE in rings obtained from LPS-treated rats, but not in those from Dex plus AG-treated endotoxemic rats. Methylene blue, an inhibitor of soluble guanylyl cyclase (GC), completely restored contractions to NE and aortic cGMP levels to normal either in LPS-treated rats or in Dex plus AG-treated endotoxemic rats, whereas the cGMP level was partially inhibited by ODQ in LPS-treated rats only. These results suggest that non-NO mediator(s) also activates soluble GC during endotoxemia. Interestingly, we found that in the presence of tetraethylammonium (an inhibitor of K⁺ channels) plus L-NAME or charybdotoxin [a specific inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels] plus ODQ, the vascular hyporeactivity to NE in the LPS-treated group was also completely restored to normal. In addition, in the presence of L-NAME or ODQ, the vascular hyporeactivity to high K⁺ was abolished in rings from the LPS-treated group. These results suggest that LPS causes the production of other mediator(s), in addition to NO, which also stimulates soluble GC (i.e., increases the formation of cGMP) and then activates the large-conductance K_Ca channels in the vascular smooth muscle causing vascular hyporeactivity.