The hippocampus is an area of the brain involved in learning and memory. It contains parallel excitatory pathways referred to as the trisynaptic pathway (which carries information as follows: entorhinal cortex -> dentate gyrus -> CA3 -> CA1 -> entorhinal cortex) and the monosynaptic pathway (entorhinal cortex -> CA1 -> entorhinal cortex). We developed a generally applicable tetanus toxin–based method for transgenic mice that permits inducible and reversible inhibition of synaptic transmission and applied it to the trisynaptic pathway while preserving transmission in the monosynaptic pathway. We found that synaptic output from CA3 in the trisynaptic pathway is dispensable and the short monosynaptic pathway is sufficient for incremental spatial learning. In contrast, the full trisynaptic pathway containing CA3 is required for rapid one-trial contextual learning, for pattern completion–based memory recall, and for spatial tuning of CA1 cells.