Ca2+ transients trigger many SNARE-dependent membrane fusion events. The homotypic fusion of yeast vacuoles occurs after a release of lumenal Ca2+. Here, we show that trans-SNARE interactions promote the release of Ca2+ from the vacuole lumen. Ypt7p-GTP, the Sec1p/Munc18-protein Vps33p, and Rho GTPases, all of which function during docking, are required for Ca2+ release. Inhibitors of SNARE function prevent Ca2+ release. Recombinant Vam7p, a soluble Q-SNARE, stimulates Ca2+ release. Vacuoles lacking either of two complementary SNAREs, Vam3p or Nyv1p, fail to release Ca2+ upon tethering. Mixing these two vacuole populations together allows Vam3p and Nyv1p to interact in trans and rescues Ca2+ release. Sec17/18p promote sustained Ca2+ release by recycling SNAREs (and perhaps other limiting factors), but are not required at the release step itself. We conclude that trans-SNARE assembly events during docking promote Ca2+ release from the vacuole lumen.