The carotid body (CB) is a chemosensory organ that detects changes in chemical composition of arterial blood and maintains homeostasis via reflex control of ventilation. Thus, in response to a fall in arterial PO2 (hypoxia), CB chemoreceptors (type I cells) depolarize, and release neurotransmitters onto afferent sensory nerve endings. Recent studies implicate ATP as a key excitatory neurotransmitter released during CB chemoexcitation, but direct evidence is lacking. Here we use the luciferin-luciferase bioluminescence assay to detect ATP, released from rat chemoreceptors in CB cultures, fresh tissue slices, and whole CB. Hypoxia evoked an increase in extracellular ATP, that was inhibited by L-type Ca2+ channel blockers and reduced by the nucleoside hydrolase, apyrase. Additionally, iberiotoxin (IbTX; 100 nM), a blocker of O-2-sensitive Ca2+-dependent K+ (BK) channels, stimulated ATP release and largely occluded the effect of hypoxia. These data strongly support a neurotransmitter role for ATP in carotid body function. (C) 2004 Elsevier Inc. All rights reserved.