The crystal structure of the ammonia transport (Amt) protein AmtB at 1.4 A resolution revealed four ammonia/ammonium (NH3/NH4+) binding sites along the similar to 20 A narrow pore. It is an open question whether the bound NH3/NH4+ are neutral (NH3) or cationic (NH4+). On the basis of the AmtB crystal structure, we calculated the pK(a) of these four NH3/NH4+ by solving the Poisson-Boltzmann equation. Except for one NH3/NH4+ binding site (Am1) at the entry point of the Amt pore, binding sites are occupied by NH3 due to lack of energy contributions from solvation, eliminating an existence of charged form NH4+ and, inevitably, its potential cation-pi interaction. The only two titratable residues in the pore, His168 and His318, are in the neutral charge state. The NH4+ charge state at the Am1 site is stabilized by Ser219 functioning as an H-bond acceptor. However, when involving explicit crystal water nearby, the NH3 charge state is stabilized by the reorientation of Ser219-OH group. This H-bond donor Ser219 significantly decreases the pK(a) of NH3/ NH4+ at the Am1 site to similar to 1. The flip/flop H-bond of Ser219 may play a dual role first in binding and subsequently in deprotonating NH4+, which is a prerequisite to conduct NH3 through the Amt pore across the membrane.