RH421 is a widely used voltage-sensitive fluorescent membrane probe. It is also photochemically reactive and its photochemistry is dependent on its solvent surroundings. The aim of this study is to use the solvent dependence of the dye's photochemistry as an indicator of its environment within a lipid membrane. It was found that the dye actually undergoes two competing photochemical reactions. One photochemical pathway is characterized by an increase in fluorescence, the other by a fluorescence decrease. The pathway followed depends on the light intensity, the excitation wavelength and the solvent. The pathway leading to a fluorescence decrease is very dependent on the polarity of the solvent. It is promoted by solvents of high polarity (e.g., methanol, ethanol and propanol), by high intensity illumination and long wavelength excitation (i.e., 546 or 577 nm). In less polar solvents (butanol, pentanol, hexanol and heptanol) the pathway leading to a fluorescence increase dominates. The photochemical behavior observed when the dye is bound to a lipid membrane was not comparable to its behavior in any of the isotropic solvents studied. This is most likely due to the anisotropic environment of the membrane, which results in a steep gradient in polarity along the length of the dye molecule.