In the current paper we describe the development of our method for quantification of underivatized individual conjugated linoleic acid (CLA) isomers by silver ion liquid chromatography (Ag⁺-HPLC). Positional and geometric CLA isomers were separated on two commercially available Chrompac (The Netherlands) ChromSpher 5 µm Lipids columns (250 x 4.6 mm) protected by a guard column (10 x 3 mm) containing the same stationary phase. Lyophilized skeletal muscle, adipose tissue, liver, brain and pancreas samples were hydrolyzed with 2M NaOH at 80-85°C for 30-35 min in sealed tubes. After cooling, the hydrolyzates were acidified with 4M HCl and the free CLA isomers were extracted with dichloromethane. The organic solvent was removed in a stream of argon and then the residue was re-dissolved in dichloromethane followed by injection of the resulting solution (10-30 µl) onto the HPLC columns (column temperature: 25°C). A mobile phase of 1.6% acetic acid and 0.0125% acetonitrile in n-hexane was chosen as the optimum mobile phase for fractionation of the individual CLA isomers. Analyses of underivatized isomers were performed by the isocratic elution program (flow-rate of 1 ml/min) and UV detection at 234 nm. All positional and geometric CLA isomers in standards and biological samples were efficiently retained on the columns and were satisfactorily separated from all endogenous species present in biological materials in about 55 min. The first low intensity peak group was trans-trans CLA isomers (23-27 min), the second highest intensity peak group was cis-trans/trans-cis isomers (31-38 min), while the last group was low intensity cis-cis isomers (41-52 min). The use of two silver ion-exchange columns with direct UV detection offers low limits of detection (0.14-1.02 ng · l^-1), quantification (0.42-3.06 ng · l^-1) and satisfactory “purity” of analytical peaks, so, this simple Ag⁺-HPLC method can be used for routine analysis of CLA isomer biodistribution in the body of animals.