We synthesized a series of potential chemoaffinity ligands for the androgen receptor (AR) by means of structural modifications of bicalutamide, a known nonsteroidal antiandrogen used in the treatment of hormone-dependent prostate cancer. We determined AR binding affinities of these ligands, identified chemoaffinity ligands by exchange assays, and confirmed irreversible binding to the AR by Scatchard analyses. AR binding affinity was determined in a competitive binding assay with a radiolabeled high-affinity AR ligand, [3H]mibolerone ([3H]MIB). For exchange assays, AR were incubated with an excess of each ligand, and then adsorbed onto hydroxyapatite (HAP). HAP-bound AR then were incubated with [3H]MIB to determine the remaining exchangeable specific binding sites. To determine the concentration of binding sites (Bmax), using Scatchard analysis, AR were incubated with a fixed concentration of ligand and increasing [3H]MIB concentrations. The ligands showed a wide range of AR binding affinities. In the exchange assays, three isothiocyanate derivatives of R-bicalutamide, the p-isothiocyanate (R-4), the p-thio-isothiocyanate (R-6), and the m-isothiocyanate (R-3), reduced exchangeable specific binding of [3H]MIB by 85, 84, and 50%, respectively. The S-isomer of p-thio-isothiocyanate (S-6), which showed 700-fold lower AR binding affinity than R-6, did not reduce exchangeable specific binding of [3H]MIB. In Scatchard analyses, the isothiocyanate derivatives R-3, R-4, and R-6 showed significant and progressive reduction in Bmax at increasing concentrations. The results indicate that initial specific reversible AR binding was required for subsequent covalent labeling, and that R-3, R-4, and R-6 bound the AR specifically and irreversibly. These isothiocyanate derivatives of R-bicalutamide are the first specific chemoaffinity ligands for the AR, and will provide valuable tools for the molecular characterization of the ligand binding domain of the AR.