ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid)), was characterized in an acetate buffer solution (pH 5.0) by cyclic voltammetry and thin layer spectroelectrochemistry. ABTS(.+) presented an electronic absorption spectrum with five maxima, at 214, 394, 414, 646 and 728 nm. There was no spectroscopic interference at 728 rim with ABTS or ABTS(2+) and was the best wavelength to measure the ABTS(.+) absorbance. Also a complete oxidation of ABTS -> ABTS(2+) produced almost a colorless solution. This spectroelectrochemical observation is novel and could be extremely useful to many investigators that study ABTS-mediated oxidations catalyzed by laccase and peroxidase. The obtained results showed that enzyme laccase (fungal source: Myceliophthora thermophila) produced a reduced ABTS and stable monocation ABTS(.+) mixture with no evidence of the dication ABTS(2+) formation. This was in agreement with the redox potential associated to this laccase. Finally the results revealed that ABTS(.+) was reduced in presence of a wide variety of organic compounds, including the interesting case of indigo blue which was completely bleached at a stoichiometric coefficient [ABTS(.+)]/[Indigo] similar to 4. These results indicate the importance of producing oxidized mediators such as ABTS(.+), either by enzymatic or electrochemical processes in order to oxidize a wide variety of refractory compounds such as aromatic dyes and polyphenolic compounds. The fact that oxidized mediators react with the final substrate alone opens new possibilities for a wide variety of electrochemical or enzymatic reactors. The mediator recycling between enzyme and substrate is a possibility that has been shown to be feasible and those results will be published elsewhere. (c) 2005 Elsevier B.V. All rights reserved.