The aim of the study was to investigate the effect of n-6 PUFA (maize silage/grass silage, soybean meal and soybean oil, control) and n-3 PUFA (grass silage, rapeseed cake and rapeseed oil, experiment) based diets on the occurrence of rumen- and duodenal digesta trans-C18:1 and CLA isomers of German Simmental bulls. The results based on rumen and duodenal digesta samples immediately taken from the bulls just after slaughter. The diet affected the occurrence of individual trans-C18:1 and CLA isomers in the rumen and duodenal digesta in different ways. The isomer trans-11,cis-13 CLA was detected as the most abundant isomer in the rumen of n-3 PUFA based diet fed bulls compared to n-6 PUFA based diet fed bulls. The trans-7,cis-9 CLA isomer was not detected in the rumen samples of bulls fed both diets, however abundant trans-7,cis-9 CLA was identified and quantified in the duodenum digesta. Both the concentration of the sum of trans-18:1 fatty acids and individual isomers in the rumen were not affected by the diet, except trans-16-18:1. The concentration of trans-16-18:1 was significantly decreased in the rumen of n-3 PUFA supplemented-fed bulls compared to n-6 PUFA supplemented-fed bulls.