Correlated spontaneous activity plays critical role in the organization of neocortical circuits during development. However, cortical mechanisms regulating activity correlation are still elusive. In this study, using two-photon calcium imaging of the barrel cortex layer 4 (L4) in living neonatal mice, we found that N-methyl-D-aspartate (NMDA) receptors (NMDARs) in L4 neurons are important for enhancement of spontaneous activity correlation. Disruption of GluN1 ( Grin1 ), an obligatory NMDAR subunit, in a sparse population of L4 neurons reduced activity correlation between GluN1 knockout (GluN1KO) neuron pairs within a barrel. This reduction in activity correlation was even detected in L4 neuron pairs in neighboring barrels, and most evident when either or both of neurons are located on the barrel edge. Our results provide evidence for the involvement of L4 neuron NMDARs in spatial organization of the spontaneous firing activity of L4 neurons in the neonatal barrel cortex. SIGNIFICANCE STATEMENT Precise ...