In this article, we examined the fluorescent properties of 4'- and 5'-aminofluorescein, unsubstituted fluorescein, and its 4'-nitro derivative in a set of solvent systems. Fluorescence lifetimes, quantum yields, time-resolved fluorescence spectra, and quantum chemical calculations allowed clarifying the reasons of the emitting properties in this dye series. In water, the dianions R2- of aminofluoresceins are practically nonfluorescent; in alcohols, the quantum yields are low. In dimethylsulfoxide (DMSO), acetonitrile, and other non-hydrogen bond donor solvents, the bright fluorescence of R2- ions is quenched either on adding small amounts of water which hydrate the carboxylate group or under conditions of protonation of this group (COO- → COOH). The last observation is possible owing to the peculiarities of the tautomerism of the 5'-aminofluorescein monoanion, HR-, which exists in DMSO as an equilibrium mixture of a colorless lactone and colored "phenolate" tautomer with an ionized xanthene moiety and unionized carboxylic group. In contrast, the R2- anion of 4'-nitrofluorescein demonstrates spectral behavior different from that of the amino derivatives. It practically does not emit in aprotic solvents; however, in alcohols or water media, its quantum yield increases to some extent. Such changing spectral properties are explained in terms of the excited-state interfragmental charge transfer.