1. Tight seal, whole-cell recordings from auditory cortex in vivo and in vitro were obtained to investigate modification of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic activity by paired-pulse afferent stimulation. 2. In recordings from urethane-anaesthetized rats (at 37 degrees C), or from cortical slices maintained in vitro (32 degrees C), afferent stimulation elicited a monosynaptic early EPSP and polysynaptic early and late IPSPs. In addition, a late EPSP could be elicited when the stimulus was preceded by an identical priming stimulus (interval approximately 200 ms). The late EPSP was attenuated by the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV, 50 microM). 3. Bath application of the gamma-aminobutyric acid-B (GABAB) receptor antagonist 3-amino-2-(4-chlorophenyl)-2-hydroxy-propylsulphonic acid (2-OH-saclofen; 50 microM) attenuated the late IPSP and clearly revealed a late EPSP. However, 2-OH-saclofen had lesser effects on the second late EPSP elicited during paired-pulse stimulation. Membrane depolarization in 2-OH-saclofen increased the magnitude of the early IPSP, which suppressed the late EPSP once again. Since pharmacological blockade of EPSPs revealed paired-pulse depression of monosynaptically elicited early and late IPSPs, these data indicate that (1) both early and late IPSPs were capable of suppressing the late EPSP, and (2) these effects were reduced during paired-pulse stimulation. 4. Pharmacological isolation of the late EPSP allowed testing of the direct effect of paired-pulse stimulation. Application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 microM), picrotoxin (10 microM) and 2-OH-saclofen (50 microM) isolated the late EPSP (onset, 3 ms; peak latency, 28 ms; peak amplitude, 7 mV; duration, 240 ms), which grew in magnitude with membrane depolarization and was largely (> 90%) blocked by APV. Paired-pulse stimulation depressed the isolated late EPSP by 30%. 5. Thus, apparent paired-pulse facilitation of the late EPSP is attributable to release from GABAergic inhibition, and not to direct facilitation. Facilitation of the late EPSP is a functional consequence of IPSP depression. The results indicate the importance of inhibition in regulating synaptic activity mediated by NMDA receptors.