Cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) was photoaffinity-labeled with a specific ligand, 3β, 5α-dihydroxycholestan-6-one (YS-64). Analysis by ODS-HPLC of peptide fragments obtained from the labeled CN-TPBP by trypsinization indicated the existence of a single specifically labeled site. Affinity gels for the purification of CN-TPBP were then prepared. As ligands for the affinity gels, 12-O-tetradecanoylphorbol 13-acetate (TPA), a typical phorbol-type tumor promoter, and benzolactam-V8-310 (BL-V8-310), a structural/biological mimic of teleocidin-class tumor promoters, were adopted. The use of these gels afforded a protein that showed a single band of 58 kDa on SDS-PAGE.