The binding mode of nicotinic agonists has been thoroughly investigated in the last decades. It is now accepted that the charged amino group is bound by a cation-π interaction to a conserved tryptophan residue, and that the aromatic moiety is projected into a hydrophobic pocket deeply located inside the binding cleft. A hydrogen bond donor/acceptor, maybe a water molecule solvating this receptor subsite, contributes to further stabilize the nicotinic ligands. The position of this water molecule has been established by several X-ray structures of the acetylcholine-binding protein. In this study, we computationally analyzed the role of this water molecule as a putative hydrogen bond donor/acceptor moiety in the agonist binding site of the three most relevant heteromeric (α4β2, α3β4) and homomeric (α7) neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Our theoretical investigation made use of epibatidine 1 and deschloroepibatidine 2 as molecular probes, and was then extended to their analogues 3 and 4, which were subsequently synthesized and tested at the three target receptor subtypes. Although the pharmacological data for the new ligands 3 and 4 indicated a reduction of the affinity at the studied nAChRs with respect to reference agonists, a variation of the selectivity profile was clearly evidenced.