Despite the wealth of data characterizing neuronal Ca2+ signals in vitro, little is known about the mechanisms of Ca2+ signaling in the intact brain. Here we used two-photon imaging to study the mechanisms underlying spontaneous and light-evoked neuronal Ca2+ signals in layer 2/3 cells of the mouse visual cortex. The Ca2+ signals were blocked by bath application (perfusing the recording chamber attached to the skull) of TTX (1 mM) as well as by the combined application of the NMDA and AMPA receptor blockers D,L-APV (100 mM) and CNQX (50 mM). Interestingly, neither bath nor local application of CNQX alone blocked the Ca2+ signals completely. Instead, the signals were entirely blocked by bath application of the NMDA receptor blockers APV or CGP (25 mM). The complete blockade was also achieved with local APV application. Furthermore, Ca2+ transients similar to the spontaneous or light-evoked signals were easily produced by local application of NMDA. Finally, the signals were largely reduced in the presence of the L-type Ca2+ channel blocker nitrendipine. Taken together our results suggest that Ca2+ signals in the visual cortex in vivo are caused by suprathreshold glutamatergic EPSPs triggering action potential firing and the activation of the voltage-gated Ca2+ channels. The postsynaptic NMDA receptor-channels seem to play the key role in this process, gating the information transfer to the neuronal elements situated downstream of layer 2/3 pyramidal cells.