Extracellular ATP and other nucleotides function as autocrine and paracrine signaling factors in many tissues. Recent studies suggest that P2 nucleotide receptors and ecto-nucleotidases compete for a limited pool of endogenously released nucleotides within cell surface microenvironments that are functionally segregated from the bulk extracellular compartment. To test this hypothesis, we have used luciferase-based methods to continuously record extracellular ATP levels in monolayers of human 1321N1 astrocytoma cells under resting conditions, during stimulation of Ca2+-mobilizing receptors for thrombin or acetylcholine, and during mechanical stimulation by hypotonic stress. Soluble luciferase was utilized as an indicator of ATP levels within the bulk extracellular compartment, whereas a chimeric protein A-luciferase, adsorbed to antibodies against a glycosylphosphatidylinositol-anchored plasma membrane protein, was used as a spatially localized probe of ATP levels at the immediate extracellular surface. Significant accumulation of ATP in the bulk extracellular compartment, under either resting (1-2 nm ATP) or stimulated (10-80 nm ATP) conditions, was observed only when endogenous ecto-ATPase activity was pharmacologically inhibited by the poorly metabolizable analog, betagamma-methylene ATP. In contrast, accumulation of submicromolar ATP in the cell surface microenvironment was readily measured even in the absence of ecto-ATPase inhibition suggesting that the spatially colocalized luciferase could effectively compete with endogenous ecto-ATPases for released ATP. Other experiments revealed a critical role for elevated cytosolic [Ca2+] in the ATP release mechanism triggered by thrombin or muscarinic receptors but not in basal ATP release or release stimulated by hypotonic stress. These observations suggest that ATP release sites are colocalized with ecto-ATPases at the astrocyte cell surface. This colocalization may act to spatially restrict the actions of released ATP as a paracrine or autocrine mediator of cell-to-cell signaling.