The cell-surface-signaling protein Notch, is required for numerous developmental processes and typically specifies which of two adjacent cells will adopt a non-neuronal developmental fate. It has recently been implicated in long-term memory formation in mammals and Drosophila. Here, we investigated whether activity-dependent synaptic plasticity at the neuromuscular junctions (NMJs) of third instar Drosophila larvae depends on Notch signaling. The length and number of axonal branches and number of presynaptic sites (boutons) in NMJ vary with the level of synaptic activity, so we increased activity at the NMJ by two complementary methods: increasing the chronic growth temperature of third instar larvae from 18 to 28 degrees C and using the double-mutant ether-a-gogo,Shaker (eagSh), both of which increase NMJ size and bouton count. Animals homozygous for the functionally null, temperature-sensitive Notch alleles, N(ts1) and N(ts2), displayed no activity-dependent increase in NMJ complexity when reared at the restrictive temperature. Dominant-negative Notch transgenic expression also blocked activity-dependent plasticity. Ectopic expression of wild-type Notch and constitutively active truncated Notch transgenes also reduced activity-dependent plasticity, suggesting that there is a "happy medium" level of Notch activity in mediating NMJ outgrowth. Last, we show that endogenous Notch is primarily expressed in the presynaptic cell bodies where its expression level is positively correlated with motor neuron activity.