Munc13-1 is a presynaptic protein with an essential role in synaptic vesicle priming. It contains a diacylglycerol (DAG)/beta phorbol ester binding C(1) domain and is a potential target of the DAG second messenger pathway that may act in parallel with PKCs. Using genetically modified mice that express a DAG/beta phorbol ester binding-deficient Munc13-1(H567K) variant instead of the wild-type protein, we determined the relative contribution of PKCs and Munc13-1 to DAG/beta phorbol ester-dependent regulation of neurotransmitter release. We show that Munc13s are the main presynaptic DAG/beta phorbol ester receptors in hippocampal neurons. Modulation of Munc13-1 activity by second messengers via the DAG/beta phorbol ester binding C(1) domain is essential for use-dependent alterations of synaptic efficacy and survival.