The ESI-formed protonated 2 -deoxycytidine, cytidine, cytarabine, and gemcitabine have been probed using infrared multiphoton dissociation (IRMPD) spectroscopy performed in the 900–2000 cm−1 region at CLIO, the Orsay Free Electron Laser facility, and in the 2800–3800 cm−1 region using a YAG-laser coupled to a table-top optical parametric oscillator/amplifier (OPO/OPA). The IRMPD spectra are compared of the protonated nucleosides with the IR spectra of their B3LYP/6-311++G(d,p)-optimized isomeric forms. The stability at room temperature of some conformers has been investigated by means of ab initiomolecular dynamics simulations. The IRMPD spectra are consistent with the formation in the ESI source of both the N3- and the O2-protonated nucleosides. The most favoured members of both families are characterized by the pyrimidine base orientedantito the furanose moiety. Concerning the O2-protonated nucleosides, IRMPD spectra and thermochemical considerations support the predominant formation of the structures with the proton oriented up relative to the furanose moiety. ©