Epibatidine is an alkaloid toxin that binds with high affinity to nicotinic and muscarinic acetylcholine receptors, and has been extensively used as a research tool. To examine binding interactions at the nicotinic receptor, it has been co-crystallised with the structural homologue acetylcholine binding protein (AChBP; PDB ID 2BYQ), and with an AChBP chimaera (3SQ6) that shares 64% sequence identity with the α7 nACh receptor. However, the binding orientations revealed by AChBP co-crystal structures may not precisely represent their receptor homologues and experimental evidence is needed to verify the ligand poses. Here we identify potential binding site interactions between epibatidine and AChBP residues, and substitute equivalent positions in the α7 nACh receptor. The effects of these are probed by [3H]epibatidine binding following the expression α7 nACh receptor cysteine mutants in HEK 293 cells. Of the sixteen mutants created, the affinity of epibatidine was unaffected by the substitutions Q55C, L106C, L116C, T146C, D160C and S162C, reduced by C186A and C187A, increased by Q114C and S144C, and abolished by W53C, Y91C, N104C, W145C, Y184C and Y191C. These results are consistent with the predicted orientations in AChBP and suggest that epibatidine is likely to occupy a similar location at α7 nACh receptors. We speculate that steric constraints placed upon the C-5 position of the pyridine ring in 3SQ6 may account for the relatively poor affinities of epibatidine derivatives that are substituted at this position.