1. Olfactory bulb neurons were dissociated from neonatal rats and plated at low density on a confluent layer of olfactory bulb astrocytes. Intracellular stimulation of presumptive mitral/tufted (M/T) cells evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in adjacent neurons. Whole-cell recording techniques and a flow-pipe drug delivery system were used to compare EPSPs with voltage-clamp recordings of currents evoked by excitatory amino acids (EAA) including N-acetylaspartylglutamate (NAAG), a putative mitral cell transmitter. 2. Cultured olfactory bulb neurons were morphologically and physiologically distinct. Large pyramidal-shaped neurons were present, which were NAAG immunoreactive; stimulation of these neurons invariably evoked EPSPs, suggesting that they were M/T cells. The majority of small bipolar neurons were glutamic acid decarboxylase (GAD) immunoreactive consistent with granule or periglomerular gamma-aminobutyric acid (GABA)ergic interneurons. 3. Monosynaptic EPSPs between M/T cells could be separated into fast and slow components by the use of EAA receptor antagonists. A fast component with a time-to-peak of 7.7 +/- 1.0 (SE) ms and half-width of 31.8 +/- 7.4 ms was blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX, 2.5 microM). The slow component (time-to-peak = 41.4 +/- 7.2 ms; half-width = 218.9 +/- 40.4 ms) was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5, 100 microM). 4. Under voltage clamp, flow-pipe applications of NAAG (10-1,000 microM) evoked inward currents at a holding potential of -60 mV in Mg-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)