Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRAB , can address this shortfall. 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRAB fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader. 2-AG and AEA increased GRAB fluorescence in N2a cells with EC values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB R) antagonist SR141617 and absent in cells expressing the mutant-GRAB . ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRAB signal within minutes primarily via activation of P2X receptors (P2X R). This response was dependent on diacylglycerol lipase β activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/β-hydrolase domain containing 6 (ABHD6). Considering that P2X R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.