Male Wistar rats received by stomach intubation 500 mg/kg of the azo dyes Sunset Yellow FCF and Orange GGN. Previous work revealed that in the Sunset Yellow FCF as well as in the Orange GGN aqueous faeces extracts a considerable orange metabolite is present, eluting as a strong peak before the unchanged dye after ion-pair reverse-phase liquid chromatography (RP-IP-HPLC). Incubation of both aqueous faeces extracts with arylsulphatase followed by high-performance liquid chromatography (HPLC) analysis, resulted in a significant orange metabolite peak height decrease together with a noticeable unchanged dye peak height increase. By means of semi-preparative HPLC, combined with cation-exchange purification, the orange Sunset Yellow FCF faeces metabolite could be isolated. It was identified by means of FT-1HMR spectroscopy as a Sunset Yellow FCF structurally related compound with the same 1HMR pattern, which is therefore modified on the free aromatic hydroxyl group