The main objective of this study was to establish the contribution of NMDA receptors to natural processing of somatosensory information within rat SI barrel cortex. Responses of 52 cells in layers I-IV of the rat barrel cortex were analyzed by PSTH (peristimulus histogram) analysis of evoked spikes in reply to brief deflections of the principal whisker in animals anesthetized with urethane. Short and longer peak latency responses within PSTHs were compared in the presence and absence of the specific NMDA and non-NMDA antagonists D(-)- 2-amino-5-phosphonovaleric acid and 6,7-dinitroquinoxaline-2,3-dione, which were administered locally to neurons by iontophoresis and additionally tested against their putative specific agonists, NMDA and quisqualate, respectively. The results suggest the following. (1) The generation of most spikes from cells in layers I-IV is dependent upon activation of NMDA receptors. However, NMDA receptors do not contribute to responses at very short latencies commensurate with monosynaptic thalamocortical relay for layer IV cells. These appear to be entirely mediated through non-NMDA receptors. (2) In the absence of transmission through NMDA receptors, non-NMDA receptors do not generate significant spike activity in later (10–100 msec latency) discharges. (3) NMDA receptor participation in first spike generation is directly dependent upon the latency of response of the cell to principal whisker deflection. (4) Latency of response, non-NMDA receptor-mediated spike generation and laminar location were powerfully covariant. (5) In addition, it was found that cells exhibiting short-duration spikes ( < 0.7 msec; “fast-spike units”) in layer IV responded powerfully at short latencies, first spikes being entirely dependent upon non-NMDA but not NMDA receptor action, later spikes (10–100 msec poststimulus) being > 80% dependent upon NMDA receptor action. It is concluded that most sensorially driven spike activity in layers I-IV is dependent upon NMDA receptor action. This appears to be enabled by contingent subthreshold depolarization largely through non-NMDA receptor action, whereas the earliest thalamocortical discharges are evoked solely through non-NMDA receptors.