The binding of L-[3H] aspartate to extensively-washed rat spinal cord synaptic membranes was investigated. Specific binding was enriched in synaptic membranes and was optimal under physiological conditions of temperature and pH. Equilibrium binding was established relatively slowly over a period of 30 min, and was totally reversible within 40 min. Saturation analysis revealed complex binding patterns. Two sites were clearly demonstrable, only one of which was shown to be saturable over the ligand concentration range employed in the study (0.1-10 microM). There was also some indication of the presence of a higher affinity site, although this was not investigated in any detail. Saturable binding demonstrated a KD = 1.4 microM and Bmax = 105 pmole/mg protein. Structure-activity studies with a range of amino acid analogues indicated that binding was stereospecific and was inhibited by a very restricted range of compounds. The most potent inhibitors of binding were L-glutamate and L-aspartate. There was no evidence for the involvement of NMDA receptors. Effects of possible endogenous modulators, including ions and guanosine nucleotides were investigated, and the chemical nature of the binding site probed with a number of protein-modifying agents.