Monoclonal anti-idiotypic antibodies specific for the combining site on a monoclonal antinicotine were used in immunocytochemistry to localize nicotine binding sites on rat brain cortical sections and in immunoaffinity chromatography to isolate receptor from solubilized brain tissue. The receptor, which consists of two subunits with Mr values of 43 and 50 kDa, was eluted from the antiidiotype column with either pH3 citrate buffer or 25 mM (-)-nicotine, but was not present in eluates from immobilized anti-Electrophorus acetylcholine receptor or anti-methotrexate. The anti-idiotypes specifically inhibited [3H]nicotine binding to rat brain homogenate and (-)-nicotine inhibited anti-idiotype binding to brain sections based on abrogation of immunofluorescence staining. These results are consistent with the operational definition of the anti-idiotypes as the internal image of nicotine, and demonstrate their value as immunochemical probes of nicotinic receptors.