Nicotinic acid-adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725-729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor-ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.