The effect of oleic (OA), linoleic (LA) and alpha-linolenic (ALA) acid used as supplements to a diet with lucerne and barley on rumen fermentation and lipid metabolism was determined in an artificial rumen. The fatty acid (FA) supplementation to a mixeddiet did not affect any parameters of rumen fermentation; the methane production was decreased nonsignificantly. The stoichiometric parameters of rumen fermentation and lipid metabolism were also affected. The concentration of total FA and proportion oflong chain fatty acids increased and proportion of medium chain fatty acids decreased. The biohydrogenation (BH) of fatty acids was characterized by nonsignificantly increased accumulation of stearic acid and trans isomers C18:1 and lower SFA/UFA ratio in the effluent. The concentrations of two main BH intermediates significantly increased compared to the control.