The pharmacology of [(125)I]-apamin binding sites was examined in rat cortical and hippocampal tissue and compared with membranes prepared from human embryonic kidney (HEK293) cells transfected with SK channel subtypes hSK1, rSK2 and rSK3. The K(D) of [(125)I]-apamin in rat cortex and hippocampus was similar to the apamin-sensitive subtypes, rSK2 and rSK3 (K(D) (pM): 6.4, 7.08, 6.56 and 8.94, respectively). In addition, [(125)I]-apamin had a K(D)=270.4pM for the putatively 'apamin-insensitive' hSK1. Apamin had about a three-fold higher affinity than [(125)I]-apamin in brain tissue and in the cells expressing the different SK channel subtypes. Pancuronium, bicuculline and d-tubocurarine displayed micromolar affinity for all five-membrane preparations, whereas dequalinium and gallamine appear to show some subtype selectivity. Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) had millimolar affinity and linopirdine had no effect. In conclusion, the pharmacology of [(125)I]-apamin binding in the cortex and hippocampus was similar to that in the apamin-sensitive clones, rSK2 and rSK3. In addition, we demonstrated low affinity [(125)I]-apamin binding for hSK1 and identified compounds that show subtype selectivity. These data cast further doubt on the identification of SK1 as encoding for the K(+) channel responsible for the apamin-insensitive sAHP.