Under conditions of collision-induced dissociation (CID), anions of α-hydroxycarboxylic acids usually fragment to yield the distinctive hydroxycarbonyl anion (m/z 45) and/or the complementary product anion formed by neutral loss of formic acid (46 u). Further support for the known two-step mechanism, involving an ion-neutral complex for the formation of the hydroxycarbonyl anion from the carboxyl group, is herein provided by tandem mass spectrometric results and density functional theory computations on the glycolate, lactate and 3-phenyllactate ions. A fourth, structurally related α-hydroxycarboxylate ion, obtained by deprotonation of mandelic acid, showed only loss of carbon dioxide upon CID. Density functional theory computations on the mandelate ion indicated that similar energy inputs were required for a direct, phenyl-assisted decarboxylation and a postulated novel rearrangement to a carbonate ester, which yielded the benzyl oxide ion upon loss of CO2. Rearrangement of the glycolate ion led to expulsion of carbon monoxide, whereas the 3-phenyllactate ion showed the loss of water and formation of the benzyl anion and the benzyl radical as competing processes. The fragmentation pathways proposed for lactate and 3-phenyllactate are supported by isotopic labeling. The relative computed energies of saddle points and product ions for all proposed fragmentation pathways are consistent with the energies supplied during CID experiments and the observed relative intensities of product ions. The diverse reaction pathways characterized for this set of four α-hydroxycarboxylate ions demonstrate that it is crucial to understand the effects of structural variations when attempting to predict the gas-phase reactivity and CID spectra of carboxylate ions. Copyright © 2013 John Wiley & Sons, Ltd.