Reverse micelles formed by ACT (Aerosol OT, sodium bis(2-ethylhexyl) sulfosuccinate) in hexane at different mole fractions of water (We) were studied with novel two-band ratiometric fluorescence probes based on 3-hydroxychromone. In the range of W-0 from 0 to 30, the intensity ratio at emission band maxima changes up to 18 times. The profile of the fluorescence response is different at low and high W-0. This suggests that at the initial step of hydration the increase of binding site polarity of the probes is accompanied by the loss of their specific interaction with ionic headgroups at the micellar interface, while at higher W-0 they respond by redistribution to apolar phase.