The synaptic vesicle protein, synaptotagmin, has been hypothesized to mediate several functions in neurotransmitter release including calcium sensing, vesicle recycling, and synaptic vesicle docking. These hypotheses are based on evidence from in vitro binding assays, peptide and antibody injection experiments, and genetic knockout studies. Synaptotagmin contains two domains that are homologous to the calcium ion (Ca2+)-binding C2 domain of protein kinase C. The two C2 domains of synaptotagmin have broadly differing ligand-binding properties. We have focused on the second C2 domain (C2B) of synaptotagmin I, in particular, on a series of conserved lysine residues on β-strand 4 of C2B. This polylysine motif binds clathrin-adapter protein AP-2, neuronal calcium channels, and inositol high polyphosphates. It also mediates Ca2+-dependent oligomerization. To investigate the importance of these lysine residues in synaptic transmission, we have introduced synaptotagmin I (syt) transgenes harboring specific polylysine motif mutations into flies otherwise lacking the synaptotagmin I protein (sytnull). Electrophysiological analyses of these mutants revealed that evoked transmitter release is decreased by ≈36% and that spontaneous release is increased approximately twofold relative to sytnull flies that express a wild type syt transgene. Synaptotagmin expression in both the mutant and the wild type transgenic lines was equivalent, as measured by semiquantitative Western blot analysis. Thus, the alteration in synaptic transmission was due to the mutation and not to the level of synaptotagmin expression. We conclude that synaptotagmin interactions mediated by the C2B polylysine motif are required to attain full synaptotagmin function in vivo. J. Comp. Neurol. 436:4–16, 2001. © 2001 Wiley-Liss, Inc.