A binary gradient elution system with photodiode array detection at 234.5 nm was used for analysis of underivatized conjugated linoleic acid (CLA). The CLA isomers were substantially retained on the columns and were distinct from saturated, unsaturated non-conjugated fatty acids, background fluctuations and endogenous substances in milk and intestinal digesta samples. All CLA isomers appeared as a pair of large peaks at 14.1 ± 0.1 and 14.6 ± 0.1 min of elution, respectively. Trace amounts of other unidentified conjugated dienes in these biological samples were detected after 18 min of elution. A ll endogenous substances in milk, meat and intestinal digesta samples are transparent in the applied UV monitoring range (234 ± 2 nm). Elimination of pre-column derivatization yields a less expensive, more specific and simpler analytical tool for routine determination of conjugated dienes in milk, meat and intestinal digesta.