The trans- as well as the cis-18:1 isomer profiles were established in cow, goat, and ewe cheese fats, with the assumption that these are representative of the corresponding milks. Argentation thin-layer chromatography was combined with low-temperature high-resolution gas-liquid chromatography on 100-m highly polar capillary columns, thus adding precision to earlier data for these species. Despite differences in the absolute content of trans-18:1 isomers between species, the relative profiles were essentially similar. Except for the minor trans delta6-delta8 group, all trans-18:1 isomers with their ethylenic bonds between positions delta4 and delta16 (including the resolved critical pair delta13/delta14) were separated and quantitated individually. As expected, vaccenic (trans delta9-18:1) acid was the main isomer, accounting for as much as 37 to 50% of the total fraction. It was observed that the goat trans-18:1 isomer profile was usually rather close to that of cows in winter (barn feeding), whereas that of the ewe shows a seasonal dependence. The trans-18:1 profile of ewe milk fats from this study resembles that of cows in the transition period between winter and summer (pasture) feeding. Regarding the cis-18:1 acid fraction, two isomers (oleic and cis-vaccenic acids) accounted for ca. 97% of that fraction for the three species, with the cis-delta12 isomer ranked third. The analytical procedure employed here appears a convenient alternative to oxidative-based procedures (generally ozonolysis), taking less time and alleviating some drawbacks of the latter procedure.