Recent crystal structures of nicotine bound to the acetylcholine binding protein (AChBP) ended a long debateconfirming that the pyridine nitrogen of nicotine is indeed hydrogen-bonded to receptor residues through abridging water molecule. Here, we describe the first direct experimental evaluation of the hydrogen-bondaffinity of the nicotinium pyridine nitrogen. The equilibrium constant of its association with a phenol is 1order of magnitude greater than the association of the acetylcholine carbonyl oxygen.