Ruminal biohydrogenation of alpha-linolenic acid is not fully understood compared with that of linoleic acid. Some hypothetical intermediates, that is, conjugated isomers of alpha-linolenic acid (cis-9,trans-11,cis-15 and cis-9,trans-13,cis-15 18:3) have never been reported to occur in ruminant fat. Therefore, milk fat was analyzed using a combination of techniques to characterize alpha-linolenic acid biohydrogenation intermediates. Tandem off-line argentation thin-layer chromatography and high-resolution gas-liquid chromatography using a 120-m highly polar, open tubular capillary column coated with 70% cyanoalkyl polysiloxane equivalent material was used for quantification. Structural characterization of fatty acids was achieved by gas-chromatography mass-spectrometry after synthesis of specific azo-derivatives. This study confirmed that minute amounts of alpha-linolenic acid biohydrogenation intermediates are present in milk fat. Routes involved in biohydrogenation of linoleic and alpha-linolenic acids in the rumen and subsequent endogenous metabolism of related biohydrogenation products are discussed.