Abstract Stepwise dissociation and tautomerism of 2,4,5,7-tetranitrofluorescein (TNF) were studied by using vis-spectroscopy in dimethylsulfoxide (DMSO), in aqueous acetone, and in cetyl-trimethylammonium chloride (CTAC) micellar solutions at ionic strength of the bulk phase 4.00 M KCl. The p K a values in DMSO and 90 mass% (CH 3) 2CO as well as the ‘apparent’ p K a a values of the substance in micellar media were determined spectrophotometrically. The neutral (molecular) form H 2R is found to be completely converted into the colorless lactone. Moreover, the lactonic structure, yellow due to ‘nitrophenolate’ absorption band, predominates also in the case of TNF dianion R 2−. Contrary to the unsubstituted fluorescein, and like 2,4,5,7-tetrabromofluorescein (eosin), the monoanion HR − of TNF with λ max 522–525 nm and E max ∼(60–62) × 10 3 dm 3 mol −1 cm −1 exists mainly as a deeply and intensively colored structure with non-ionized carboxylic and ionized hydroxylic group; its fluorescence spectra in various media are registered. In 90% acetone, the Stokes shift is 1.17 × 10 3 cm −1, fluorescence lifetime equals 2.3 ns. An extremely expressed trend to dianion–lactone formation of R 2− ion of TNF is confirmed in the systems studied. For TNF in DMSO, in aqueous acetone, in surfactant micelles, and in trichloromethane extracts of ionic associatiates with N( n-Bu) 4 + and N( n-Hept) 4 +, the deeply colored ‘quinon–phenolate’ dianion, typical for all hydroxyxanthenes, is not registered at all. The sequence of dissociation of functional groups in solution is confirmed using IR spectroscopy in DMSO.