Acetylcholine (ACh) has been considered an important excitatory neurotransmitter in the carotid body (CB). Its physiological and pharmacological effects, metabolism, release, and receptors have been well documented in several species. Various nicotinic and muscarinic ACh receptors are present in both afferent nerve endings and glomus cells. Therefore, ACh can depolarize or hyperpolarize the cell membrane depending on the available receptor type in the vicinity. Binding of ACh to its receptor can create a wide variety of cellular responses including opening cation channels (nicotinic ACh receptor activation), releasing Ca(2+) from intracellular storage sites (via muscarinic ACh receptors), and modulating activities of K(+) and Ca(2+) channels. Interactions between ACh and other neurotransmitters (dopamine, adenosine, nitric oxide) have been known, and they may induce complicated responses. Cholinergic biology in the CB differs among species and even within the same species due to different genetic composition. Development and environment influence cholinergic biology. We discuss these issues in light of current knowledge of neuroscience.