We have combined millisecond activation of channelrhodopsin-2 (ChR2), a light-gated ion channel, with two-photon calcium imaging to investigate active synaptic contacts in rat hippocampal slice cultures. Calcium influx was larger during light-induced action potentials than during action potentials induced by somatic current injection, leading to highly reproducible synaptic transmission. Pairing of light stimulation with postsynaptic depolarization induced long-term potentiation, making this technique ideal for genetic and pharmacological dissection of synaptic plasticity.