This is the first report of the application of silver-ion impregnated high-performance liquid chromatography (Ag(+)-HPLC) to the separation of complex mixtures of conjugated linolenic acid (CLA) isomers present in commercial CLA sources and foods and in biological specimens. This method showed a clear separation of CLA isomers into three groups related to their trans,trans, cis,trans or trans,cis, and cic,cis configuration of the conjugated double-bond system. In addition, this method separated individual positional isomers of the conjugated diene system within each geometrical isomeric group. Following Ag(+)-HPLC isolation, gas chromatography (GC)-electron impact mass spectrometry, and GC-direct deposition-Fourier transformed infrared spectroscopy were used to confirm the identity of two major positional isomers in the cis/trans region, i.e., delta 8,10- and delta 11,13-octadecadienoic acids, which had not been chromatographically resolved previously. Furthermore, the potential of this method was demonstrated by showing different Ag(+)-HPLC profiles exhibiting patterns of isomeric distributions for biological specimens from animals fed a diet containing a commercial CLA preparation, as well as for a commercial cheese product.