[3H]Ethynylbicycloorthobenzoate ([3H]EBOB) is a recently developed specific radioligand for the noncompetitive blocker site of the gamma-aminobutyric acid (GABA) receptor, which is the target of many polychlorocycloalkane and heterocyclic insecticides. This study characterizes [3H]EBOB binding and the action of insecticides on the GABA-gated chloride channel of cerebellar granule neurons in primary culture. Specific [3H]EBOB binding increases rapidly between days 3 and 7 and then remains constant for days 7 to 12 in culture. A single, high-affinity and saturable binding site is involved with Kd = 0.51 nM and Bmax = 239 fmol/mg protein. GABA and muscimol are 6- to 12-fold more potent as inhibitors of [3H]EBOB binding to membranes from cultured cerebellar granule neurons than from cerebellar tissue, possibly because of localization of specific subunits in the granule cells. GABA-stimulated 36Cl- influx is also inhibited by seven insecticidal compounds (including EBOB, 12-ketoendrin and alpha-endosulfan) as well as picrotoxinin and t-butylbicyclophosphorothionate with IC50 levels ranging from 290 to 15300 nM, which correlate well (r = 0.92) with their inhibitory potencies for [3H]EBOB binding (1 to 773 nM). These findings validate the use of [3H]EBOB binding in cultured cerebellar granule cells for pharmacological studies on insecticides and other toxicants acting at the noncompetitive blocker or coupled site of the GABAA receptor/chloride channel complex.