A method is presented for identification of positional isomers of dichlorinated fatty acids, based on derivatization to picolinyl esters prior to gas chromatographic/mass spectrometric analysis in the electron ionization mode. The mass spectra of the picolinyl esters showed structure-specific fragmentation patterns. By using the picolinyl ester, 5,6-dichlorotetradecanoic acid was identified as a metabolite from a cell-culture medium obtained by culturing human cell lines in media supplemented with threo-9,10-dichlorooctadecanoic acid. This indicates that dichlorinated fatty acids are degraded by beta-oxidation. It is also possible to locate tentatively the position of chlorine atoms in 5,6-dichlorotetradecanoic acid as its methyl ester or pyrrolidide.