AbstractInositol 1,4,5‐trisphosphate (InsP3) receptors are calcium‐release channels found in the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane of diverse cell types. InsP3receptors release Ca2+from ER/SR lumenal stores in response to InsP3generated from various stimuli. The complex spatial and temporal patterns of InsP3receptor‐mediated Ca2+release regulate many cellular processes, ranging from gene transcription to memory. Ankyrins are adaptor proteins implicated in the targeting of ion channels and transporters to specialized membrane domains. Multiple independent studies have documented in vitro and in vivo interactions between ankyrin polypeptides and the InsP3receptor. Moreover, loss of ankyrin‐B leads to loss of InsP3receptor membrane expression and stability in cardiomyocytes. Despite extensive biochemical and functional data, the validity of in vivo ankyrin–InsP3receptor interactions remains controversial. This controversy is based on inconsistencies between a previously identified ankyrin‐binding region on the InsP3receptor and InsP3receptor topology data that demonstrate the inaccessibility of this lumenal binding site on the InsP3receptor to cytosolic ankyrin polypeptides. Here we use two methods to revisit the requirements on InsP3receptor for ankyrin binding. We demonstrate that ankyrin‐B interacts with the cytoplasmic N‐terminal domain of InsP3receptor. In summary, our findings demonstrate that the ankyrin‐binding site is located on the cytoplasmic face of the InsP3receptor, thus validating the feasibility of in vivo ankyrin–InsP3receptor interactions. J. Cell. Biochem. 104: 1244–1253, 2008. © 2008 Wiley‐Liss, Inc.