Within the course of this spectroscopic research, we revealed novel features of the protolytic behavior, which extend the knowledge of the chemistry of xanthene dyes and rationalize the utilization of these compounds. In addition to the well-known tautomerism of the molecular form, H2R, of fluorescein dyes, new aspects of tautomeric transformation of anions are disclosed. First, for the dyes bearing the substituents in the phthalic acid residue, 4'- and 5'-aminofluoresceins and 4'-fluorescein isothiocyanate, the monoanion HR- exists in non hydrogen bond donor-solvents not only as a tautomer with the ionized carboxylic and non-ionized OH group, but also as a 'phenolate' ion with non-ionized COOH group. Such state of HR- ions is typical for dyes bearing halogen atoms or NO2 groups in the xanthenes moiety, but was not observed until now in the case of substitution in the phthalic residue. Second, the possibility of existence of the HR- species in DMSO in form of colorless lactone is deduced for the 5'-aminofluorescein using the visible and IR spectra. This results in a dramatic difference in medium effects. For instance, whereas for fluorescein in DMSO, the inversion of the stepwise ionization constants takes place and the value equals 0.08, the same ratio for 5'-aminofluorescein is as high as 800. In addition, the values of sulfonefluorescein, erythrosin, methyl ether of fluorescein, and phenol red were obtained in order to verify the acidity scale in DMSO and to support the detailed scheme of protolytic equilibria of fluorescein dyes.