Sunset Yellow FCF, Orange GGN and 1-amino-2-naphthol-6-sulphonic acid (ANSA) were administered to male Wistar rats by stomach intubation. Aromatic sulphonic acids excreted in 24-h urines after enzymatic cleavage of the azo link in the dyes were isolated using a thoroughly elaborated ion-pair extraction method and further separated by means of a reversed-phase ion-pair liquid chromatographic system. Blank 24-h rat urines were extracted and run at the same time under identical analytical circumstances. In addition to the peaks corresponding to sulphanilic and metanilic acid and their N-acetylated derivatives, which are metabolites arising from Sunset Yellow FCF and Orange GGN, respectively, an important common peak appeared on the chromatograms, which was absent from the blank urine extracts. After analysis of 24-h urine of rats that had received ANSA, a peak with the same retention time as this unknown common peak could be detected under the same liquid chromatographic conditions, the retention time of which was different from that of the ANSA standard. However, after derivatization of the ANSA standard with acetic anhydride, followed by liquid chromatographic examination of the derivatised mixture, two peaks appeared on the chromatogram, the first of which had the same retention time and the same UV-spectrum as the unknown common peak in rat urine extracts. By means of semi-preparative liquid chromatographic separation and isolation, followed by further purification over a cation-exchange resin, the compound corresponding to the unknown common peak could be identified by FT-PMR spectroscopy as N-acetyl-ANSA