Unimolecular phototautomeric reactions in 4-thiouracil, 1-methyl-4-thiouracil and 6-aza-4-thiouracil were studied using the matrix-isolation technique combined with infrared absorption spectroscopy. For monomers of these compounds, isolated in solid argon at 10 K, an intramolecular proton-transfer photoreaction was observed. Upon UV (λ > 345 nm) irradiation, the initial oxo–thione forms of 4-thiouracils were converted into the corresponding oxo–thiol tautomers. The photogenerated oxo–thiol isomers were identified by comparing their experimental IR spectra with the spectra theoretically calculated at the DFT(B3LYP)/6-311 G(2d,p) level. Good agreement between the observed and predicted pattern of spectral bands allowed a reliable identification. This is the first report on experimental observation of isomeric forms of 4-thiouracils other than the canonical oxo–thione tautomers.