In vitro anaerobic incubations were used to determine the effect of different oils (LO-linseed, SO-sunflower, FO-fish oil) on trans fatty acid production in rumen fluid and to test if combining of monensin (MON) with the oils affects the interactions on trans fatty acid concentrations in mixed cultures of ruminal microorganisms. Two different sources of rumen fluid were used; the inoculum from the sheep fed hay and barley (80:20%)--the inoculum A and the inoculum from the sheep fed alfalfa and barley (80:20 %)--the inoculum B. The analyses showed that inoculum B contained more short chain fatty acids (SCFA), medium chain fatty acids (MCFA) and saturated fatty acids (SFA) than inoculum A. In contrast, inoculum A contained more unsaturated fatty acids (UFA) than inoculum B. The results show, that the oils affected the biohydrogenation of fatty acids (FA) by increasing the concentration of C18:0 (3-7 times) and trans C18:1 isomers (2-9 times). The concentration of two main intermediates of FA biohydrogenation-- cis 9, trans 11 C18:2 (CLA) and trans 11C18:1 (TVA) were increased with the oils, but FO was more efficient than other plant oils on CLA and TVA production. The monensin treatment had similar effect on FA metabolism as the oil treatment in comparison to unincubated control. The interactions of monensin treatment with the oils were characterized with decrease (LO+MON, SO+MON) or increase (FO+MON) of the proportions of C18:0 and trans C18:1 isomers in comparison to oil treatment. The highest concentrations of two main isomers--CLA,TVA were found in the samples containing fish oil and monensin. In conclusion, fish oil treatment and monensin with fish oil treatment was more efficient than other plant oils in the effect on trans fatty acid production (mainly CLA and TVA) in fermentation fluid in vitro.