The objectives of this study were to characterize the ontogeny of the L-glutamate (glutamate) and N-methyl-D-aspartate (NMDA) binding sites in the developing guinea pig hippocampus, and to determine the effect of acute in vitro ethanol exposure on these binding sites. Specific [)H]glutamate binding and NMDA-sensitive [H-3]glutamate binding were determined using a guinea pig hippocampal synaptic membrane preparation (HSMP). To characterize the ontogeny of the density (B-max) and affinity (K-d) of the glutamate and NMDA binding sites, saturation analysis was conducted on HSMP of guinea pigs at gestational day (GD) 50 (immature fetus; term, GD 68), GD 62 (mature, near-term fetus), postnatal day (PD) 13 (neonate), and PD > 60(adult). To examine the effect of ethanol on the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 50, GD 62, PD 13, and PD > 60 was incubated with ethanol (0-100 mM), followed by determination of specific [H-3]glutamate binding and NMDA-sensitive [H-3]glutamate binding. To determine the effect of 50 mM ethanol on the B-max and K-d of the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 62 and PD > 60 was incubated with 0 or 50 mM ethanol followed by saturation analysis. The B-max values of the hippocampal glutamate and NMDA binding sites were greater at GD 62 and PD 13 compared with GD 50 and PD > 60, but there was no change in the K-d of the binding sites throughout development. Ethanol did not alter hippocampal specific [H-3]glutamate binding or NMDA-sensitive [H-3]glutamate binding at any of the ages studied, and did not alter the hippocampal B-max or K-d of the glutamate or NMDA binding sites at GD 62 and PD > 60.