The triplet states of unsubstituted and 5-substituted derivatives of salicylic acid and methyl salicylate, some of which are used as matrices in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of macromolecules, were studied in acetonitrile and cyclohexane by transient absorption and time-resolved luminescence spectroscopy. The results suggest a tautomeric structure of the triplet states of salicylic acid as well as that of its methyl ester and its 5-hydroxy- and 5-methoxy-substituted derivatives. In this tautomeric structure, the ortho-hydroxy hydrogen has been transferred to the carbonyl oxygen. No differences were observed between the triplet-triplet absorption spectra of the acids and the corresponding methyl esters. For the 5-hydroxy-and 5-methoxy-compounds, evidence for long-lived phototautomers was found. The P-type delayed fluorescence of methyl salicylate is consistent with the known tautomer fluorescence at 440 nm, implying a tautomeric structure of the triplet state. Similarly, for the 5-methoxy-substituted compound a unique delayed fluorescence spectrum, red-shifted relative to the prompt fluorescence spectrum, was observed and attributed to excimer or tautomer fluorescence, The results presented here contrast with previous reports on the absence of intramolecular hydrogen atom transfer on the singlet surface of the MALDI matrices 5-methoxy-and 5-hydroxysalicylic acid and their respective methyl esters, The resulting tautomers and their reaction products may be relevant for analyte ionization in MALDI.