Synaptotagmin is an integral synaptic vesicle protein proposed to be involved in Ca(2+)-dependent exocytosis during synaptic transmission. Null mutations in synaptotagmin have been made in Drosophila, and the protein's in vivo function has been assayed at the neuromuscular synapse. In the absence of synaptotagmin, synaptic transmission is dramatically impaired but is not abolished. In null mutants, evoked vesicle release is decreased by a factor of 10. Moreover, the fidelity of excitation-secretion coupling is impaired so that a given stimulus generates a more variable amount of secretion. However, this residual evoked release shows Ca(2+)-dependence similar to normal release, suggesting either that synaptotagmin is not the Ca2+ sensor or that a second, independent Ca2+ sensor exists. While evoked transmission is suppressed, the rate of spontaneous vesicle fusion is increased by a factor of 5. We conclude that synaptotagmin is not an absolutely essential component of the Ca(2+)-dependent secretion pathway in synaptic transmission but is necessary for normal levels of transmission. Our data support a model in which synaptotagmin functions as a negative regulator of spontaneous vesicle fusion and acts to increase the efficiency of excitation-secretion coupling during synaptic transmission.