We have examined the interaction of the neutral and protonated species of the local anesthetic prilocaine (PLC) with phosphatidylcholine (PC) bilayers combining experimental (H-1 NMR) and theoretical (molecular dynamics simulations) approaches. DOSY experiments allowed the determination of the association constants of protonated (K-a = 9 L/mol) and neutral (K-a = 21 L/mol) PLC to egg PC liposomes. Saturation transfer difference (STD) experiments showed a different trend depending on pH: At high pH the PLC hydrogen saturation was essentially uniform and at pH 5.5 the experiments show an enhancement of the aromatic moiety hydrogen saturation, with respect to the tail. Molecular dynamics simulations, performed with PLC molecules on planar bilayers of palmitoyloleyl-PC, revealed a preferential orientation for the protonated PLC species at the polar interface of the bilayer, and a nonoriented and deeper insertion for neutral PLC. Such preferential location of protonated and neutral PLC inside the bilayer can be described as different transient sites which could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel, justifying differences in the anesthetic's potency upon ionization.