Deactivation of N-methyl-D-aspartate (NMDA) channels after brief agonist exposure determines the duration of their synaptic activation during excitatory neurotransmission. We performed patch-clamp recordings of L-glutamate responses from human embryonic kidney tumoral cells (HEK293) expressing NR1 subunit variants lacking exon 5 together with the NR2B subunit. These responses had deactivation components that lasted several seconds. The presence of exon 5 or spermine greatly accelerated deactivation of L-glutamate responses through alterations in desensitization. These effects were also observed at positive holding potentials and in the presence of physiological Mg(2+). Thus NR1 splicing and polyamines may have profound effects on the kinetics of NMDA receptor-mediated synaptic transmission.