Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca2+ from intracellular acidic Ca2+ stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca2+ channels that release organellar Ca2+ in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP (32P-5N3NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca2+ signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N3-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca2+ release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family. Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) activates two-pore channels (TPCs) to release Ca2+ from intracellular acidic Ca2+ stores. Results: A photoactivatable probe based on NAADP labels proteins distinct from TPCs. Conclusion: NAADP may bind to an accessory protein within a larger TPC complex. Significance: First evidence that TPCs act as NAADP-activated Ca2+ release channels, but not NAADP receptors.