The binding of [ 3 H]ryanodine was determined in microsomal membrane preparations obtained from cultured cerebellar granule cells. A K D of 1 nM and a B max of 64 fmol/mg protein were calculated from saturation experiments. This binding was calcium dependent and maximum values were obtained at 100-300 mM Ca 11 . Caffeine increased [ 3 H]ryanodine binding only at Ca 11 concentrations lower than optimum. The binding of [ 3 H]ryanodine was inhibited by ruthenium red, procaine and the d-isomer of hexachlorocyclohexane (d-HCH). Dantrolene, a ryanodine receptor antagonist in skeletal sarcoplasmic reticulum, and the pesticide g-HCH (lindane) had no effect on [ 3 H]ryanodine binding. The obtained binding parameters, the Ca 11 dependence and the effects of the agents tested agree with previous reports using brain microsomal membranes, further indicating a neuronal localization of [ 3 H]ryanodine binding sites. When the interaction between dantrolene and gand d-HCH was tested, no changes were detected on the effects of HCH isomers on [ 3 H]ryanodine binding. Dantrolene, which inhibits Ca 11 release from sarcoplasmic reticulum and from unidentified internal Ca 11 stores in neurons, also inhibits the intracellular Ca 11 mobilization induced by g-HCH but only marginally that induced by d-HCH in the same preparation of cerebellar granule cells (Rosa et al.; Toxicol Appl Pharmacol, in press). Thus, the results obtained in this work verify the presence of different intracellular sites of action for the two HCH isomers: the ryanodine Ca 11 channel for d-HCH and an unidentified dantrolene-sensitive Ca 11 channel for the g-HCH isomer.