In ruminants, cis and trans C18:1 isomers are intermediates of fatty acid transformations in the rumen and their relative amounts shape the nutritional quality of ruminant products. However, their exact synthetic pathways are unclear and their proportions change with the forage:concentrate ratio in ruminant diets. This study traced the metabolism of vaccenic acid, the main trans C18:1 isomer found in the rumen, through the incubation of labeled vaccenic acid with mixed ruminal microbes adapted to different diets. [1-13C]trans-11 C18:1 was added to in vitro cultures with ruminal fluids of sheep fed either a forage or a concentrate diet. 13C enrichment in fatty acids was analyzed by gas-chromatography-mass spectrometry after 0, 5 and 24 h of incubation. 13C enrichment was found in stearic acid and in all cis and trans C18:1 isomers. Amounts of 13C found in fatty acids showed that 95% of vaccenic acid was saturated to stearic acid after 5 h of incubation with the concentrate diet, against 78% with the forage diet. We conclude that most vaccenic acid is saturated to stearic acid, but some is isomerized to all cis and trans C18:1 isomers, with probably more isomerization in sheep fed a forage diet.