Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ store and the presence of IP3 receptor (IP3R)/Ca2+ channel on the secretory granule membrane have been established, the identity of the IP3R types present in the secretory granules are not known. We have therefore investigated the presence of different types of IP3R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy, and found the existence of all three types of IP3R in the secretory granules. To determine whether these IP3Rs interact with CGA and CGB, each IP3R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells and the expressed IP3R type and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP3R form complexes with CGA and CGB in the cells. To further confirm whether the IP3R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP3R and CGA and CGB was tested by co-immunoprecipitation experiements of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP3R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP3R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca2+-binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol Ca2+/mol with a dissociation constant (Kd) of 1.5 mM at pH 5.5 but virtually no Ca2+ at pH 7.5. The high capacity, low affinity Ca2+-binding property of CGB at pH 5.5 is comparable to that of CGA, and is in line with its role as a Ca2+ storage protein in the secretory granules.