To examine how functional circuits are established in the brain, we studied excitatory transmission in early postnatal hippocampus. Spontaneous neural activity was sufficient to selectively deliver GluR4-containing AMPA receptors (AMPA-Rs) into synapses. This delivery allowed non-functional connections to transmit at resting potentials and required NMDA receptors (NMDA-Rs) but not CaMKII activation. Subsequently, these delivered receptors were exchanged with non-synaptic GluR2-containing AMPA-Rs in a manner requiring little neuronal activity. The enhanced transmission resulting from this delivery and subsequent exchange was maintained for at least several days and required an interaction between GluR2 and NSF. Thus, this sequence of subunit-specific trafficking events triggered by spontaneous activity in early postnatal development may be crucial for initial establishment of long-lasting functional circuitry.