Ratiometric fluorescent probes based on 3-hydroxyflavone (3HF) are highly sensitive tools for studying polarity, hydration, electronic polarizability, and electrostatics in different microheterogeneous systems, including protein molecules. In the present work, a reactive derivative of 3HF, 6-bromomethyl-4'-diethylamino-3-hydroxyflavone, recently synthesized in our group, was applied to label covalently bovine lens alpha-crystallin. The labeling of SH and NH(2) groups are clearly distinguished by spectroscopic criteria. We observe that the NH(2) labeling creates the positive charge in the proximity to fluorophore, which results in strong internal Stark effect producing the shift in excitation spectrum by ca. 15 nm. Analysis of excitation-dependent fluorescence spectra allows separation of the emission profiles of these SH- and NH(2)-labeled species. Applying recently developed multiparametric analysis of the obtained emission spectra, we described the physicochemical properties of the sites of SH and NH(2) labeling in alpha-crystallin. The site of SH labeling has medium-low polarity (dielectric constant, epsilon = 4.9 +/- 0.9) is protic, and does not contain proximal aromatic residues (according to the obtained refractive index, n = 1.41 +/- 0.14). The site of NH(2) labeling is also of medium-low polarity. The novel label due to its two-wavelength ratiometric response and high sensitivity to the type of labeling may offer new possibilities in the studies of structure, dynamics, and interactions of proteins by probing their SH- and NH(2)-labeling sites.