Several compounds, such as epibatidine, A-85380, and their analogs, have been identified recently as nAChR ligands whose affinities lie in the low picomolar range. Accurate measurement of such high affinities is fraught with certain technical difficulties, which may account for the inconsistency of previously reported affinities of epibatidine, ranging from 4 to 60 pM. Here, we demonstrate that (+/-)-[3H]epibatidine (1-500 pM) binds to a single population of sites in rat brain with KD of 8 +/- 2 pM. This affinity was confirmed in both kinetic experiments and competition assays with (+/-)-[3H]epibatidine and (-)-[3H]cytisine, which were performed under experimental conditions developed specifically for ligands with subnanomolar affinities. Variations from these conditions decreased the observed affinities.