Prototropic equilibria in 4-thiouracil has been investigated by high-level ab initio calculations. Six of the most stable tautomers have been studied at various levels of theory including the MP4(SDQ)/6-311G(2d,2p)//MP2/6-31G(d,p) level. At all levels of theory, the 2-keto-4-thio form is shown to be the most stable one while the 4-thio-2-enolic form is predicted to be the second most stable tautomer. Aqueous solvation is shown only to destabilize the minor tautomers with an energy difference between the two most stable tautomers estimated to be 12.02 kcal/mol, while the corresponding difference in the gas phase is 10.4 kcal/mol. At MP4/6-311G(2d,2p)//MP2/6-31G(d,p) in the gas phase, the following order of stability is observed; 4TU1 > 4TU2 > 4TU3 > 4TU4 > 4TU5 > 4TU6, while in an aqueous phase a stability order of 4TU1 > 4TU2 > 4TU4 > 4TU3 > 4TU6 > 4TU5 is predicted. We corroborate our theoretical findings with spectroscopic studies of UV absorption and the luminescence spectra. Although the UV absorption spectrum of the neutral keto-thionic forms of 4-thiouracil in an aqueous medium is essentially a single-component one indicating that 4-thiouracil exists as a single tautomer in aqueous medium, the luminescence spectrum of these compounds in an ethanol solution was found to be a two-component one suggesting that ethanol could play a key role in stabilizing the minor tautomeric species in excited states. Using combined experimental and theoretical results, we conclude that in an aqueous medium, 4-thiouracil exists in the 2-keto-4-thio form and excludes the possible existence of minor tautomers whereas in ethanol it could exist in two tautomeric forms.