Cultured spinal cord networks grown on microelectrode arrays display complex patterns of spontaneous burst and spike activity. During disinhibition with bicuculline and strychnine, synchronized burst patterns routinely emerge. However, the variability of both intra- and interculture burst periods and durations are typically large under these conditions. As a further step in simplification of synaptic interactions, we blocked excitatory AMPA synapses with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX), resulting in network activity mediated through the N-methyl-D-aspartate (NMDA) receptor (NMDA(ONLY)). This activity was APV sensitive. The oscillation under NMDA(ONLY) conditions at 37 degrees C was characterized by a period of 2.9 +/- 0.3 s (16 separate cultures). More than 98% of all neurons recorded participated in this highly rhythmic activity. The temporal coefficients of variation, reflecting the rhythmic nature of the oscillation, were 3.7, 4.7, and 4.9% for burst rate, burst duration, and interburst interval, respectively [mean coefficients of variation (CVs) for 16 cultures]. The oscillation persisted for at least 12 h without change (maximum observation time). Once established, it was not perturbed by agents that block mGlu receptors, GABA(B) receptors, cholinergic receptors, purinergic receptors, tachykinin receptors, serotonin (5-HT) receptors, dopamine receptors, electrical synapses, burst afterhyperpolarization, NMDA receptor desensitization, or the hyperpolarization-activated current. However, the oscillation was destroyed by bath application of NMDA (20-50 microM). These results suggest a presynaptic mechanism underlying this periodic rhythm that is solely dependent on the NMDA synapse. When the AMPA/kainate synapse was the sole driving force (n = 6), the resulting burst patterns showed much higher variability and did not develop the highly periodic, synchronized nature of the NMDA(ONLY) activity. Network size or age did not appear to influence the reliability of expression of the NMDA(ONLY) activity pattern. For this reason, we suggest that the NMDA(ONLY) condition unmasks a fundamental rhythmogenic mechanism of possible functional importance during periods of NMDA receptor-dominated activity, such as embryonic and early postnatal development.