AbstractWe propose an instrumental method based on liquid chromatography coupled to negative ion‐spray ionization (ISP(−)) tandem mass spectrometry (MS/MS), for the simultaneous analysis of eight hydroxylated brominated diphenyl ethers (OH‐PBDEs) in four different environmental matrices (soil, fish, sludge and particulate matter). The reversed‐phase chromatographic separation was performed on a 50 mm Xbridge C18 column, and the compounds were well resolved with a gradient consisting of a ternary mixture of 5 mMammonium acetate, methanol and acetonitrile. Detection was performed in the multiple reaction monitoring (MRM) mode using the [M − H]− ion as base peak. The fragmentation pathways of the OH‐PBDEs varied according to the hydroxyl substitution in the benzene rings and produced characteristic MRM transitions needed for resolving isomeric compounds. The method is acceptable for quantification in the high picogram per gram dry weight (dw) level for all matrices analyzed. Repeatability and reproducibility tested at 75 pg µl−1 were below 10% using internal standard quantification. The ISP (−) enhancement due to matrix effects was in the 76–132% range and the highest values corresponded to sludge samples. The use of the proposed method based on LC‐ISP(−)‐MS/MS opens a new way to directly determine OH‐PBDEs without the need of derivatization. Copyright © 2007 John Wiley & Sons, Ltd.