Several new derivatives of fluorescein were synthesized. The dyes were characterized by NMR; and the absorbance, excitation, and emission spectra were measured. The fluorescence quantum yields of the dyes were determined. The pKa3 values of the dyes were measured by fluorescence titration. The characteristics of the fluorescein and sulfonefluorescein derivatives were compared. The most promising dye for use in cell analysis appeared to be compound 9, which was given the name Vita Blue. The dibutyrate ester of Vita Blue was made and the compound was given the name Vita Blue dibutyrate (VBDB, 14). The K, of VBDB with pig liver esterase was measured and found to be 4 x M. The pKa3 of Vita Blue was 7.56 f 0.03; both acidic and basic forms were fluorescent (dual fluorescence). The use of VBDB as an intramolecular esterase substrate and its utility for the discrimination between live and dead cells by flow cytometry is described.Key terms: Viable dye, fluorescent esterase substrate, flow cytometry, dual fluorescence, new xanthene dyes Xanthene dyes are among the most commonly used class of fluorescent dyes. Fluorescein (11, a dihydroxy xanthene dye, is a particularly useful dye for biological purposes for several reasons: the dianionic form of the dye is very soluble in water; its pKa3 is 6.3-6.7 and it is therefore mostly deprotonated and fluorescent at physiological pH (7.0-7.6); the excitation maximum of the dye (490 nm) is well matched to the 488-nm line of the argon ion laser; it has a high extinction coefficient (8 x lo4 M-lcm-l) and a high quantum yield (0.92). The last three properties of the dye allow the fI uorescence detection of minute quantities of the dye. In addition, derivatives of fluorescein are readily synthesized and allow utilization of the dye for biological purposes; e.g., fluorescein isothiocyanate can be used to make fluorescent derivatives of proteins (3), and fluorescein diacetate has utility as a membrane permeable esterase substrate (15).Our objective was to synthesize new fluorescent dyes for use in flow cytometry with all the desirable characteristics of fluorescein described above, but with excitation maxima to the red of fluorescein. The advantages of a dye with longer wavelength excitation and emission are several: background fluorescence from biological material (DNA, proteins) diminishes, the cost and size of the laser diminishes, and the possibility of dual laser excitation using an argon ion laser as the lower wavelength laser arises.We were interested in examining the properties of sulfonefluorescein and sulfonenaphthofluorescein derivatives as well as fluorescein and naphthofluorescein derivatives. In general, the sulfonefluorescein analogs are more difficult to synthesize than the fluorescein analogs due to the deactivating character of sulfonic acid groups in Friedel-Crafts acylations; this difficulty in synthesis may explain why these dyes have not been previously exploited. For example, there are only a handful of references to sulfonefluorescein in the literature as compare...