The biological characteristics of the globular substance, a precursor of otoconia, are unclear. In the present study, the ATP-induced internal free Ca²⁺ concentration ([Ca²⁺]_i) changes of the globular substance and the ATP distribution in the vestibular organ were investigated using a Ca²⁺ indicator, fluo 3, and an adenine nucleotide-specific fluorochrome, quinacrine, by means of confocal laser scanning microscopy. [Ca²⁺]_i showed a rapid and dose-dependent increase in response to ATP with a 50% effective concentration (EC₅₀) of 16.7 µM. This reaction was independent of external Ca²⁺, indicating the presence of an internal Ca²⁺ reservoir. Neither adenosine, ,-methylene-ATP, 3'-O-(4-benzoylbenzoyl)-ATP, ADP, nor UTP evoked this reaction, whereas 2-methylthio-ATP induced an increase of [Ca²⁺]_i with an EC₅₀ of 14.4 µM. Moreover, P2 antagonists, reactive blue 2 and suramin, and a phospholipase C inhibitor, U-73122, inhibited the ATP-induced [Ca²⁺]_i increase. These findings indicate the presence of a P2Y purinoceptor on the globular substance. In addition, granular fluorescence was observed in the quinacrine-stained macular sensory epithelium, indicating the presence of ATP-containing granules in this tissue. These results suggest that a paracrine mechanism involving ATP may exist in the macula and that this mechanism regulates the biological behavior of the globular substance.