Affinity probes for the noncompetitive blocker or picrotoxinin site of the gamma-aminobutyric acid (GABA)-gated chloride channel were designed for four types of applications: photoaffinity reagents to covalently label the binding site; fluorescent probes for receptor analysis; biotinylated compounds and agarose/sepharose conjugates for affinity chromatography; ligand-protein/ enzyme conjugates for immunoassay. These 5e-tert-butyl-2e-[4-(substituted-ethynyl) phenyl]-1,3-dithianes were optimized by structure-activity studies for potency as inhibitors of 3H ethynylbicycloorthobenzoate binding to bovine brain membranes, measured as the concentration for 50% inhibition (IC50). Preferred compounds are 5e-(CH3)3CCH(CH2S)2CH-2e-C6H6-4-C = CCH2OCH2C(O)R, wherein R confers the following properties and IC50 values: R = SCH2CH2SCH2C(O)C6H4-4-N3, photoaffinity, 9 nM; R = NHCH2CH2NHC(O)C6H2-2-OH,5-1,4-N3, photoaffinity, 105 nM: R = SCH2CH2S-4-benzofurazan-7-NO2, fluorescent, 13 nM: R = SCH2CH2SCH2-5-fluorescein, fluorescent, 27 nM; R = NHCH2CH2NH[C(O)(CH2)5NH]2-biotin, affinity chromatography, 190 nM. The most potent photoaffinity ligand (IC50 9 nM) was labeled at 7 Ci mmol-1 by reacting the appropriate thiol with 3H 4-azidophenacyl bromide (obtained by alumina-catalyzed tritium exchange of its enolizable hydrogens). The first steps have been taken in using the NCB site for affinity chromatography of the GABAA receptor in CHAPS-solubilized bovine brain membranes with the dithiane-biotin probe and an avidin-acrylic bead system or with an analogous dithiane-agarose/ sepharose column eluting with GABA or dithiane as above (R = OH). A protein conjugate of a related dithiane-monosulfone elicited production of specific antisera in rabbits. These findings illustrate the diversity and utility of new affinity probes prepared in the alkynylphenyldithiane series.