Structures of the solvation shells of O-protonated formamide (H(+)FA) have been characterized by infrared spectroscopy and ab initio calculations at varying stages of hydration. A few isomeric structures, including both H(+)FA- and H3O+-centered forms, were identified from a close examination of the hydrogen-bonded and non-hydrogen-bonded NH and OH stretching spectra of H(+)FA(H2O)(n) (n = 3 and 4) produced by a supersonic expansion. Theoretical investigations of H(+)FA(H2O)(1-3) indicate that filling of the first hydration shell of the protonated formamide cation with three water molecules is not an energetically favorable process. The process is hampered by the hydrogen bond anticooperative effect, which prohibits both NH bonds to be involved in hydrogen bonding. Further increasing the cluster size to n = 4 results in grouping of the water molecules on one side of the formamide, rather than forming a filled first solvation shell of H(+)FA. The grouping, in effect, enhances the proton affinity of the water molecule in direct contact with the excess proton and progressively moves th proton away from H(+)FA, producing an H3O+ ion core. Detailed analysis of the results, both qualitatively and quantitatively, reveals the existence of a low-barrier proton transfer process, which is expected to occur similarly in aqueous solutions 'during acid-catalyzed amide hydrolysis. In this work, we also successfully identified a shell-filled isomer for H+(FA)(3)H2O for the first time. It is demonstrated that a combined investigation of cluster ions by infrared spectroscopy and ab initio calculations allows for an unambiguous determination of both the protonation sites and hydration structures of biomolecules in the gas phase.