2-Acetyl-2-decarboxamido-oxytetracycline (ADOTC) is a major impurity of oxytetracycline (OTC) produced as a side product during fermentation. ADOTC was isolated from OTC and other impurities using preparative HPLC. The preparative column was an Xterra MS. C-18 chromatographic column (100 mm x 19 turn W., 5 mum), and the mobile phase contained methanol-water (27:73 (v/v)) with 0.08 M formic acid added. The flow rate was 9.0 ml/min. It was possible to isolate few milligram ADOTC in a day. The compound was unambiguously identified using NMR and MS-MS. The anti-microbial activity against activated sludge bacteria was deter-mined giving a potency of only 3% of that of OTC. With tetracycline-resistant bacteria, no anti-microbial activity was observed, indicating a mode of action similar to that of OTC.