Mammalian homologues of Drosophila Trp form plasma membrane channels that mediate Ca(2+) influx in response to activation of phospholipase C and internal Ca(2+) store depletion. Previous studies showed that human Trp3 is activated by inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and identified interacting domains, one on Trp and two on IP(3)R. We now find that Trp3 binds Ca(2+)-calmodulin (Ca(2+)/CaM) at a site that overlaps with the IP(3)R binding domain. Using patch-clamp recordings from inside-out patches, we further show that Trp3 has a high intrinsic activity that is suppressed by Ca(2+)/CaM under resting conditions, and that Trp3 is activated by the following: a Trp-binding peptide from IP(3)R that displaces CaM from Trp3, a myosin light chain kinase Ca(2+)/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca(2+)/CaM. We conclude that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP(3)Rs.