In the neocortex, fast excitatory synaptic transmission can typically be blocked by using excitatory amino acid (EAA) receptor antagonists. In recordings from layer II/III neocortical pyramidal neurons, we observed an evoked excitatory postsynaptic potential (EPSP) or current (EPSC) in the presence of EAA receptor antagonists (40-100 μM D-APV+20 μM CNQX, or 5 mM kynurenic acid) plus the GABAA-receptor antagonist bicuculline (BIC, 20 μM). This EAA-antagonist resistant EPSC was observed in about 70% of neurons tested. It had a duration of approximately 20 ms and an amplitude of 61.5±6.8 pA at -70 mV (n=35). The EAA-antagonist resistant EPSC current-voltage relation was linear and reversed near 0 mV (n=23). The nonselective nicotinic acetylcholine receptor (nAChR) antagonists dihydro-β-erythroidine (DHβE, 100 μM) or mecamylamine (50 μM) reduced EPSC amplitudes by 42 (n=20) and 33% (n=9), respectively. EPSC kinetics were not significantly changed by either antagonist. Bath application of 10 μM neostigmine, a potent acetylcholinesterase inhibitor, prolonged the EPSC decay time. EAA-antagonist resistant EPSCs were observed in the presence of antagonists of metabotropic glutamate, serotonergic (5-HT3) and purinergic (P2) receptors. The EAA-antagonist resistant EPSC appears to be due in part to activation of postsynaptic nAChRs. These results suggest the existence of functional synaptic nAChRs on pyramidal neurons in rat neocortex. © 2000 Elsevier Science B.V.