Inositol triphosphate (IP3) receptors, are commonly responsible for release of Ca2+ from intracellular stores. IP3 binding to type 1, but not type 3, IP3 receptors is inhibited by calmodulin in a Ca2+-independent fashion. Using HSAB-calmodulin to photoaffinity label GST-fusion proteins representing different sequences of the type 1 IP3 receptor, I identified a region that binds calmodulin in both the presence and absence of Ca2+. This region contains a residue (Ser1589) that is phosphorylated by protein kinase A, but protein kinase A-catalysed phosphorylation of purified type 1 IP3 receptors was unaffected by calmodulin in either the presence or absence of Ca2+. I also demonstrated that IP3-evoked Ca2+ release from cerebellar microsomes and cell types that express predominantly type 1, 2 or 3 IP3 receptors, is similarly susceptible to inhibition by calmodulin. In SH-SY5Y cells, which express largely type 1 IP3 receptors, calmodulin inhibited IP3-evoked Ca2+ mobilization only in the presence of Ca2+. This effect of calmodulin did not result from enhanced metabolism of IP3 since calmodulin also reduced the sensitivity of Ca2+ stores to adenophostin A, a non-metabolizable agonist of IP3 receptors. Thus, in contrast to the inhibition of IP3 binding by calmodulin, inhibition of IP3-evoked Ca2+-release occurs only in the presence of Ca2+ and appears to affect all three subtypes of IP3 receptor. Calmodulin also interacts with type 1 IP3 receptors in the absence of Ca2+, but the effects of this interaction on IP3 receptor function remain to be determined.