The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical. But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive' as no final Ca2+-dependent triggering step has been observed. Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events. Membrane fusion involves several subreactions: priming, tethering and docking. Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown. Here we show that Ca2+ is released from the vacuolar lumen following completion of the docking step. We have identified calmodulin as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion. Calmodulin binds tightly to vacuoles upon Ca2+ release. Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing. Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes. We propose that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions. However, the origin of the Ca2+ signal, its receptor and its mode of processing differ.