An immunosorbent containing antiisoproturon and antichlortoluron antibodies immobilised on aldehyde-activated silica was employed for the on-line preconcentration and liquid chromatography–diode array detection of several phenylureas. The efficiency of the coupling of the immunosorbent to the liquid chromatographic system and the characteristics of the immunosorbent were evaluated. The on-line system allowed the selective trace enrichment of chlortoluron, isoproturon, metobromuron, linuron and chlorbromuron at the 0.05–0.5 µg l–1 level in ground and river waters and provided detection limits in the range 0.01–0.03µg l–1 by percolating only 10 ml of water sample. The proposed method was validated by analysing freeze-dried tap water samples with a high content in pesticides of different chemical functionalities. The results obtained were compared with the mean value obtained in an interlaboratory exercise.