Spaced patterns of repetitive synaptic activation often result in a long-lasting, protein synthesis-dependent potentiation of synaptic transmission, known as late-phase long-term potentiation (L-LTP) that may serve as a substrate for long-term memory. Behavioral studies showed that posttraining blockade of NMDA subtype of the glutamate receptor (NMDAR) impaired long-term memory, although NMDAR activation is generally known to be required during LTP induction. In this study, we found that the establishment of L-LTP in vivo requires NMDAR activation within a critical time window after LTP induction. In the developing visual system of Xenopus laevis tadpole, L-LTP of retinotectal synapses could be induced by three episodes of theta burst stimulation (TBS) of the optic nerve with 5 min spacing ("spaced TBS"), but not by three TBS episodes applied en masse or spaced with intervals ≥10 min. Within a time window of ∼30 min after the spaced TBS, local perfusion of the tectum with NMDAR antagonist d-AP5 or Ca(2+)-chelator EGTA-AM impaired the establishment of L-LTP, indicating the requirement of postinduction activation of NMDAR/Ca(2+) signaling. Moreover, inhibiting spontaneous spiking activity in the tectum by local application of tetrodotoxin (TTX) prevented L-LTP when TTX was applied for 15 min immediately after the spaced TBS but not 1 h later, whereas the same postinduction TTX application in the retina had no effect. These findings offer new insights into the synaptic basis for the requirement of postlearning activation of NMDARs and point to the importance of postlearning spontaneous circuit activity in memory formation.