Abstract A method based on gas chromatography (GC) separation followed by ion trap tandem mass spectrometry detection in EI mode (ITD‐MS/MS), using isotope dilution, was developed for the determination of ten native polybrominated diphenyl ethers (PBDEs) and four 13 C 12 ‐labeled congeners in biological (fat tissue and human serum) and food samples. The highest‐mass fragment ions were used as precursor ions for those congeners with molecular ions with m/z values higher than the maximum of the instrument. In these cases (hepta‐BDEs and 13 C 12 ‐hexa‐BDEs) no fragmentation was achieved under the experimental conditions employed. Repeatability (lower than 9%) and reproducibility (lower than 13%), expressed as relative standard deviation (RSD, n = 3 and 4, respectively), were satisfactory. Similarly, the coefficient of variation (n = 4) of the isotopic ratio between the two most abundant product ions was lower than 10 and 6% for native and labeled congeners, respectively. To evaluate the feasibility of the method, the optimized isotope dilution GC/ITD‐MS/MS method was used for the quantitation of selected PBDE congeners in different samples including adipose tissue, human serum and foodstuff samples, from three inter‐laboratory comparative exercises, covering a wide range of concentrations. A solid‐phase extraction procedure, previously developed for PCB determination in small‐size bird serum samples, was successfully applied to quantification of PBDEs in 1 mL samples of human serum. Copyright © 2005 John Wiley & Sons, Ltd.