ATP is emerging as an ubiquitous extracellular messenger. However, measurement of ATP concentrations in the pericellular space is problematic. To this aim, we have engineered a firefly luciferase-folate receptor chimeric protein that retains the N-terminal leader sequence and the C-terminal GPI anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase (pmeLUC), is targeted and localized to the outer aspect of the plasma membrane. PmeLUC is sensitive to ATP in the low micromolar to millimolar level and is insensitive to all other nucleotides. To identify pathways for nonlytic ATP release, we transfected pmeLUC into cells expressing the recombinant or native P2X₇ receptor (P2X₇R). Both cell types release large amounts of ATP (100–200 μM) in response to P2X₇R activation. This novel approach unveils a hitherto unsuspected nonlytic pathway for the release of large amounts of ATP that might contribute to spreading activation and recruitment of immune cells at inflammatory sites.