Carbon monoxide (CO) is proposed as a physiological messenger. CO activates cGMP and has a direct effect on potassium channels. Both actions of CO lead to hyperpolarization of a cell's resting membrane potential, suggesting that CO may function as a hyperpolarizing factor, although direct evidence is still lacking. Here we take advantage of the known membrane potential gradient that exists in the muscle layers of the gastrointestinal tract to determine whether CO is an endogenous hyperpolarizing factor. We find that heme oxygenase-2-null mice have depolarized smooth muscle cells and that the membrane potential gradient in the gut is abolished. Exogenous CO hyperpolarizes the membrane potential. Regions of the canine gastrointestinal tract that are more hyperpolarized generate more CO and have higher heme oxygenase activity than more depolarized regions. Our results suggest that CO is a critical hyperpolarizing factor required for the maintenance of intestinal smooth muscle membrane potential and gradient.A role for carbon monoxide (CO) is proposed in cell signaling and neurotransmission (1, 2), hormone release (3, 4), as a cytoprotective agent (5), and in regulation of vascular tone (6, 7). The major source of CO is from the breakdown of heme by heme oxygenase (HO); the absence of HO-2, the constitutive HO isoform, significantly impairs the production of CO in gut and other tissues (8). HO-2 is widely expressed in tissues containing smooth muscle, including blood vessels (6), the pulmonary system (9), and the gastrointestinal tract (10), suggesting that CO is continually produced in these tissues. Multiple mechanisms of action for CO have been proposed, including activation of guanylyl cyclase and direct activation of K ϩ channels (11). Both mechanisms can result in membrane hyperpolarization, raising the possibility that CO can act as an endogenous hyperpolarizing factor. To determine whether CO is an endogenous hyperpolarizing factor, we took advantage of the known membrane potential gradients that exist in the muscle layers of the gastrointestinal tract. We chose the gastrointestinal tract as our model because our previous studies in the HO-2-null mouse demonstrate that intestinal smooth muscle is depolarized in the HO-2-null mouse (2), and because of the presence of a measurable smooth muscle membrane potential gradient that has not been reported in vascular smooth muscle. We hypothesized that if CO acts as a physiologically relevant hyperpolarizing factor, then CO production should mirror the membrane potential gradient along the long axis of the stomach as well as across the gut wall and that these resting membrane potential gradients would be diminished or abolished in HO-2-null mice. In the gastrointestinal tract of all species studied, there is a large gradient (Ϸ30 mV) in membrane potential along the long axis of the stomach from the fundus (proximal stomach) to the pylorus (distal stomach) (12). There is also a membrane potential gradient across the thickness of circular muscle layer (12, 13). In the ...