Intercellular glial Ca2+ waves constitute a signaling pathway between glial cells. Artificial stimuli have previously been used to evoke these waves, and their physiological significance has been questioned. We report here that Ca2+ waves occur spontaneously in rat retinal glial cells, both in the isolated retina and in vivo. These spontaneous waves are propagated by ATP release. In the isolated retina, suramin (P2 receptor antagonist) reduces the frequency of spontaneous wave generation by 53%, and apyrase (ATP-hydrolyzing enzyme) reduces frequency by 95-100%. Luciferin-luciferase chemiluminescence reveals waves of ATP matching the spontaneous Ca2+ waves, indicating that ATP release occurs as spontaneous Ca2+ waves are generated. Wave generation also depends on age. Spontaneous wave frequency rises from 0.27 to 1.0 per minute per mm2, as rats age from 20 to 120 d. The sensitivity of glia to ATP does not increase with age, but the ATP released by evoked waves is 31% greater in 120-d-old than in 20-d-old rats, suggesting that increased ATP release in older animals could account for the higher frequency of wave generation. Simultaneous imaging of glial Ca 2+ and arterioles in the isolated retina demonstrates that spontaneous waves alter vessel diameter, implying that spontaneous waves may have a significant impact on retinal physiology. Spontaneous intercellular glial Ca2+ waves also occur in the retina in vivo, with frequency, speed, and diameter similar to the isolated retina. Increased spontaneous wave occurrence with age suggests that wave generation may be related to retinal pathology.