[3H]Cis-4-phosphonomethyl-2-piperidine carboxylic acid ([3H]CGS 19755) was used to investigate the pharmacology and characteristics of the N-methyl-D-aspartate (NMDA) receptor recognition site from Triton X-100-treated membranes of rat spinal cord and cerebral cortex. The association of [3H]CGS 19755 was biphasic in both spinal cord and cerebral cortical membranes reaching a maximum after 5 min of incubation then decreasing to a steady level after an additional 10 min, suggesting that a proportion of the binding is unstable. The dissociation of the stable binding component was biphasic with rate constants at 4 degrees C of 1.55 and 0.020 min-1 for the spinal cord and 1.48 and 0.051 min-1 for the cerebral cortex. These multiple sites could not be captured in the saturation studies which were best fitted to a one-site model using non-linear regression analysis. Depending on the time of incubation with [3H]CGS 19755, KD and Bmax values differed; 9.9-26.1 nM and 25-96 fmol/mg protein vs 14.0-26.5 nM and 449-900 fmol/mg protein for spinal cord and cerebral cortex, respectively. The rank order of potency of inhibiting [3H]CGS 19755 binding was similar in both tissues: L-glutamate > CGS 19755 = CPP > NMDA. The specific NMDA agonist cis-2,4-methanoglutamate potently inhibited [3H]CGS 19755 binding as did MDL 100,925, although the latter was one order of magnitude less potent in the spinal cord than in the brain. The Hill coefficients were significantly lower than unity. In both tissues, AMPA, kainate and glycine competed poorly with [3H]CGS 19755.(ABSTRACT TRUNCATED AT 250 WORDS)