The feasibility of using a silica-based immunosorbent for the selective on-line preconcentration of several phenylurea pesticides in drinking and surface waters was demonstrated. The immunosorbent was packed in a precolumn which was used in the same way as a conventional C,, or PLRP-S precolumn: percolation of aqueous samples through the precolumn, then direct coupling to a C,, analytical column and desorption using an acetonitrile gradient from 20 to 80% acetonitrile. Although the retention of the different phenylureas was based on antigen-antibody interactions in the precolumn and on hydrophobic interactions in the analytical column, the efficiency of the on-line system was excellent as shown by the on-line preconcentration and analysis of 12 phenylureas of different polarities. The characteristics of the immunosorbent (cross-reactivity, breakthrough of analytes, capacity, selectivity and reusability) were discussed. An application to the analysis of surface waters showed the high selectivity of this sorbent. Detection limits from a 50-ml sample were ranging between 0.05 and 0.5 pg/l for 10 phenylureas in a mixture of 13. An application to the rapid monitoring of isoproturon in the river Seine was also reported.