Twenty-two substituted 7-hydroxycoumarins were studied by negative ion electrospray ionization collision- induced dissociation (CID) mass spectrometry. Fragmentation pathways were also investigated by computation method using the B3LYP density functional theory. In general, the most important fragmentations of the 7- hydroxycoumarin [M - H](-) ions were the elimination of CO(2) and CO which agreed with the calculated energies of the proposed fragmentation reactions. In most cases, methyl group elimination was also favorable. Methyl group elimination occurred in three different ways, the most interesting being hydrogen rearrangement from a neighboring alkyl group to a ring carbon, which led to a benzyl radical formation. In some cases, CH(2)CO elimination was observed as well. Isomeric compounds gave rise to different CID spectra.