The fragmentation reactions of the molecular ion of the three isomeric methylpyridines (2-picoline 1, 3-picoline 2, and 4-picoline 3) were studied using methods of tandem mass spectrometry and quantum chemical calculations with Gaussian-3 (G3) theory using the B3LYP density functional method (G3//B3LYP). Additionally RRKM calculations of the microcanonical rate constants of selected reaction pathways were performed. Derivatives of 2- and 4-picoline which bear an isotopically labeled methyl substituent (CD3: 1-d3 and 3-d3, 13CH3: 1-13C and 3-13C) were also analyzed. Of the four primary dissociation reactions of the picoline radical cations 1+–3+, the loss of a hydrogen atom, the elimination of an ethyne molecule and the elimination of a hydrogen cyanide molecule are observed in the MIKE spectra, while the loss of a methyl radical is only found in the collisional activation (CA) mass spectra of the molecular ions. The CA mass spectra of the labeled picoline radical ions reveal that some positional hydrogen exchange and carbon exchange takes place before the loss of the methyl radical. According to the G3 and RRKM calculations, the direct cleavage of the methyl substituent competes effectively with increasing internal energy of the ion with the loss of a methyl radical after rearrangements. The other three pathways found for metastable molecular ions are accompanied by efficient or complete hydrogen scrambling. The theoretical calculations prove that this scrambling occurs mainly by initial H-transfer from the methyl group to the pyridine ring and a subsequent H-ring walk. The calculations show further, that loss of H may occur from many of the intermediate ions after the rearrangements but that the direct loss of H from the methyl substituent competes successfully even at low energies, contrary to the behavior of the toluene radical ion. The elimination of an ethyne molecule gives rise to a broad flat-topped signal in the mass-analyzed ion kinetic energy (MIKE) spectra, and the MIKE spectra of 1-13C and 3-13C exhibit a 1:1 sharing of the 13C-label between ionic and neutral products. These particular features are explained by the theoretical calculations by a first rearrangement of the picoline radical cations to a seven-membered aza-cycloheptatriene radical cation 4+ followed by a rearrangement to a more rigid bicyclo-[3.2.0] isomer before the elimination of ethyne. Finally, the elimination of a hydrogen cyanide molecule exhibits only minor loss of 13C-label in the case of 1-13C and 3-13C. This observation does not agree with any mechanism including a rearrangement to 4+. The theoretical calculations suggest that instead the loss of the hydrogen cyanide molecule proceeds by a cleavage of the pyridine ring after the initial H-transfer from the methyl substituent. Although this pathway needs more energy than a rearrangement to 4+, its rate coefficients are superior owing to its loose transition states.