Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed byASE, were 85% for C12BAC and 79% for C14BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 microg/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water.