Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into increased action potential (AP) activity on the sinus nerve, which increases the drive to breathe. The mechanism by which AP activity increases is unresolved, but acetylcholine (ACh), acting through nicotinic receptors, is postulated to be a major contributor to nerve excitation based partly on the demonstration that pharmacological antagonism of nicotinic receptors reduces the afferent nerve response in some studies. However, most previous studies relied on indirect measures of chemoreceptor activity or utilized a recording configuration that is sensitive to AP morphology in addition to AP frequency. In the present study, single-unit AP activity was recorded from the soma of rat chemoreceptor neurons in vitro. The nicotinic blocker mecamylamine (50 microM) ablated the excitatory actions of exogenous ACh and increased, rather than decreased, AP activity during moderate hypoxia. At higher dosage (500 microM) AP height was reduced, conduction velocity slowed, and conduction failure occurred, especially during hypoxia, producing the appearance of a decreased response to hypoxia. Recovery from mecamylamine block was slow (>10 min). In contrast to mecamylamine, suramin, a P2X receptor blocker, reversibly inhibited the response to hypoxia, suggesting relatively free diffusion of drugs to the glomus cell/nerve synaptic site. These results strongly suggest that ACh acting through nicotinic receptors does not mediate excitatory transmission in rat carotid body and that previous results demonstrating such a role may have been partially influenced by changes in AP morphology or conduction failure.