Binding of [125I]-labelled ifenprodil, a non-competitive N-methyl-D-aspartate (NMDA) antagonist acting at the polyamine domain, was studied in washed, frozen-thawed synaptic membranes. Under these conditions where the NMDA channel is essentially in a closed channel state and in the presence of GBR 12909, [125I]ifenprodil binding was rapid, reversible, stereospecific, saturable and to a single population of sites (Kd 76 microM, Bmax 140 nmol/mg protein). Binding was inhibited by spermine, spermidine and ifenprodil congeners. These characteristics differed from those found in fresh membranes (open channel state), with ifenprodil congeners being less potent and potencies of polyamines being unchanged. These data suggest independent, but interacting sites for polyamines and ifenprodil congeners, the latter sensitive to endogenous modulators, labelled by [125I]ifenprodil and probably not NMDA-linked. High affinity binding of ifenprodil congeners seems likely to require an open ('activated') NMDA channel.