In the present study, we combined a powerful and novel receptor binding data analysis technique. Fourier-derived affinity spectrum analysis (FASA), with the nonlinear regression analysis program LIGAND to resolve benzodiazepine receptor heterogeneity in rat spinal cord. With FASA, we identified three distinct [3H]Ro15-1788 binding populations: two high-affinity sites (0.4 and 5 nM) for the radioligand and a lower-affinity site (150 nM) that is insensitive to the imidazopyridine alpidem. With the affinities for the radioligand determined with FASA, the Ki values of 13 competing ligands were calculated with LIGAND. All of the ligands studied displayed the highest affinity for site 1 (the highest-affinity [3H]Ro15-1788 binding site), with the exception of AHR 11797. Site 2 had high affinity for Ro15-1788, lower affinity for flunitrazepam and beta-CCM and very low, but measurable, affinity for zolpidem. The alpidem-insensitive binding site, studied in isolation by performing competitive binding assays in the presence of 65 microM alpidem, showed relatively low affinity for all of the ligands studied, and its physiological relevance is not yet known.