In the present report we have reevaluated specific [3H]resiniferatoxin (RTX) binding, thought to represent the vanilloid (capsaicin) receptor, to whole spinal cord and dorsal horn membranes of the pig using a modified [3H]RTX binding assay. The high nonspecific [3H]RTX binding of the original protocol was reduced by the addition of alpha 1-acid glycoprotein (AGP), a plasma protein that binds RTX, to the assay mixture after the binding reaction had been terminated. Specific [3H]RTX binding to pig whole spinal cord and dorsal horn membranes followed sigmoidal saturation kinetics indicating apparent positive cooperativity. The cooperativity index determined by fitting the data to the Hill equation was 2.31 +/- 0.24 in the spinal cord and 2.27 +/- 0.13 in the dorsal horn. The apparent dissociation constants in spinal cord and dorsal horn membranes were 87.8 +/- 2.7 and 103.9 +/- 1.9 pM; the receptor densities were 23 +/- 3 and 203 +/- 5 fmol/mg protein, respectively. In parallel experiments, rat spinal cord membranes bound [3H]RTX with 2 - 3 fold higher affinity, equal positive cooperativity, and a 49 +/- 6 fmol/mg receptor density. As predicted by the modified Hill equation, at low receptor occupancy nonradioactive RTX produced biphasic competition curves. Capsaicin and the competitive antagonist capsazepine also fully displaced specifically bound [3H]RTX from pig dorsal horn membranes with Ki values of 9.7 +/- 1.7 microM and 6.8 +/- 0.7 microM, respectively; the corresponding Hill coefficients were 1.81 +/- 0.17 and 2.32 +/- 0.11. [3H]RTX binding was not inhibited by resiniferonol 9, 13, 14-orthophenylacetate, the biologically inactive parent diterpene of RTX. These findings suggest that the vanilloid receptor present in the dorsal horn of the pig, like those present in human and in the rat, is a receptor cluster in which the subunits cooperate.