Synaptotagmins (Syts) are transmembrane proteins with two Ca²⁺-binding C₂ domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca²⁺ sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379–388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290–18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca²⁺ concentrations ([Ca²⁺]) below 10 μM, whereas other isoforms require 200–500 μM [Ca²⁺] or show no Ca²⁺-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594–599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1–5 μM [Ca²⁺] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93–104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C₂A domain of Syt VII inhibit Ca²⁺-triggered secretion of β-hexosaminidase and surface translocation of Lgp120, whereas the C₂A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C₂A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca²⁺-dependent lysosome exocytosis.