Theoretical estimations at the B3LYP/aug-cc-pVTZ/B3LYP/6-31+G(d) level for the 298 K gas-phase acidities (GPAs) of all the possible deprotonation sites of the most stable tautomers of uracil, thymine, and cytosine are reported. The gas-phase acidities of the nucleobases estimated by direct calculations and derived from isodesmic reactions are very similar. For uracil, the GPAs of the N1, N3; C5, and C6 sites are 332.8 +/- 2.2, 345.1 +/- 2.2, 380.2 +/- 0.5, and 366.2 +/- 0.5 kcal/mol, respectively. For thymine, the GPAs of the N1, N3, C-5-CH3, and C6 sites are 334.5 +/- 2.2, 345.7 +/- 2.2, 376.1 +/- 2.1, and 367.5 +/- 0.5 kcal/mol, respectively. For keto cytosine, the GPAs of N1, N4-H-b, N4-H-a, C5, and C6 are 345.6 +/- 2.2, 348.7 +/- 2.1, 353.5 +/- 2.1, 379.3 +/- 0.5, and 371.2 0.5 kcal/mol, respectively. For trans-enol cytosine, the GPAs of the hydroxyl group, N4-H-b, N4-H-a, C5, and C6 are 347.2 +/- 2.1, 351.9 +/- 2.1, 355.4 +/- 2.1, 386.4 +/- 0.5, and 393.8 +/- 0.5 kcal/mol, respectively. For uracil, thymine, and keto cytosine, N1 is the most acidic site while C5 is the least acidic one. For enol cytosine, the most acidic site is the hydroxyl group and the least acidic site is C6. On the basis of these results, uracil and thymine are more acidic than cytosine. The acidities of the keto and trans-enol cytosine are approximately equal.