The photoreaction of indigo and two other derivatives in its reduced (leuco) form was investigated by absorption and fluorescence (steady-state and time-resolved) techniques. The fluorescence quantum yield (ϕF) dependence with the UV irradiation time was found to increase up to a value of ϕF ≈ 0.2−0.3 (after 16 min) for indigo and ϕF = 0.2 (at ∼150 min) for its derivative 4,4′-dibutoxy-7,7′-dimethoxy- 5,5′-dinitroindigo (DBMNI). With a model compound, where rotation around the central C−C bond is blocked, the ϕF value was found constant with the UV irradiation time. Timeresolved fluorescence revealed that initially the decays are fitted with a biexponential law (with 0.12 and 2.17 ns), ending with an almost monoexponential decay (∼2.17 ns). Quantum yields for the isomerization photoreaction (ϕR) were also obtained for indigo and DBMNI with values of 0.9 and 0.007, respectively. The results are rationalized in terms of a photoisomerization (conversion) reaction occurring in the first excited singlet state of trans to cis forms of leuco indigo.