Synapses can be temporarily strengthened by bursts of action potentials, which are thought to be a central aspect of information processing in the brain. This study provides evidence that protein kinase C (PKC)-dependent phosphorylation of synaptotagmin-1 is an essential step in this strengthening. A mutation that prevents synaptotagmin-1 phosphorylation abolishes this strengthening, both after action potential bursts and upon direct PKC activation by a synthetic analog of diacylglycerol, whereas basal synaptic transmission is unaffected. This suggests that synaptotagmin-1 acts in a cooperative fashion with Munc18-1 and Munc13-1, which were previously identified as essential diacylglycerol/PKC substrates. Together these data identify a central pathway linking bursts of action potentials to enhanced synaptic strength.