Abstract The behavior of coumarin 343 (C343), a common molecular probe utilized in solvation dynamics experiments, was studied in water/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/benzene reverse micelles (RMs). In all the studies performed until now, C343 was not soluble in the organic solvent used to create the RM systems (namely different alkanes such as n-heptane, cyclohexane or isooctane). In this work we have chosen benzene as the organic solvent because C343 is completely soluble at the work concentration used (∼10−6M). Thus, a well known AOT RM system: benzene/AOT/water has been used in order to gain insights, for the first time, on how the RMs’ formation can affect the C343 spectroscopic behavior. Dissolved in pure benzene C343 exists as a dimer since the intermolecular H-bond interactions are very strong in this solvent. When introduced to the AOT RMs, C343 resides in the RMs’ interface and, at low occupation number the probability of finding more than one C343 molecule is reduced and only C343 monomer species is detected. From the spectral changes it was possible to determine the critical micelle concentration at W0=0 and 10 and the C343 partition constants between two pseudophases. In summary, in this work we have shown how the spectroscopic behavior of C343 is dramatically altered because of the partition of the dye to the AOT RMs’ interfaces.