The influence of adding selenite (Se^IV) or selenate (Se^VI) to ovine ruminal fluid containing linoleic acid (LA) on the profile of fatty acids, especially conjugated linoleic acid (CLA) isomers and their metabolites was investigated. Dietary LA is incorporated by rumen bacteria, isomerized to other geometric and positional isomers, metabolized into CLA isomers, biohydrogenated to trans-vaccenic acid (TVA) and finally to C18:0. Considering the above, ovine ruminal fluid was incubated in vitro at 39°C under CO₂ either alone (the control ruminal fluid) or with a combination of LA (1.67 mg/ml), a low (0.167 μg/ml) or high (1.67 μg/ml) level of selenium as Se^IV or Se^VI. Tubes with examined ruminal fluid were removed after 0, 6, 12, 18, and 24 hrs of incubation and then submitted for determination of fatty acids (FA). FA, as methyl esters, were quantitated using capillary gas chromatography and flame-ionization detection. Both concentrations of Se^IV added to the ruminal fluid with LA usually decreased the concentrations of individual CLA isomers, especially cis9trans11CLA (c9t11CLA) and the sum of all CLA isomers in the ruminal fluid in comparison with the fluid containing only LA. Our studies documented that Se^IV reduced the capacity of bacterial isomerase, which turns the cis9-bond into a trans10-bond. The addition of Se^IV to the ruminal fluid with LA decreased the concentration of TVA compared with the fluid with only LA; a decrease in the loss of TVA was observed with increasing concentrations of Se^IV. The presence of Se^IV in the ovine fluid with LA stimulated the biohydrogenation of TVA to C18:0. The addition of LA to the incubated fluid, irrespectively of the presence of Se^IV, increased the concentration of C20:5n-3. Se^VI in the ruminal fluid with LA usually more efficiently increased the concentration of c9t11CLA, t10c12CLA, c9c11CLA and t9t11CLA, from 6 until 24 hrs of incubation compared with the fluid containing LA, regardless of the presence of Se^IV. The concentration of TVA in the fluid containing Se^VI and LA is higher than in the fluid with Se^IV and LA. Se^VI in the fluid increased the concentration of C18:0. As a consequence, Se^VI added to the fluid increased the yield of final biohydrogenation to C18:0 compared with the fluid with LA, irrespective of the presence of Se^VI. Further studies are required to clarify the effects of other Se-compounds and fatty acids on concentrations of fatty acids, especially CLA isomers and their precursors, in the ruminal fluid.