Four Hereford steers (500 +/- 4.5 kg of BW) cannulated in the proximal duodenum were used to evaluate the effects of vegetable oil source or fish oil quantity on ruminal biohydrogenation (BH) and CLA outflow. Steers were fed 1 of 4 treatment diets in a 2 x 2 factorial arrangement of treatments (oil source: canola vs. corn oil; fish oil quantity: 0 or 1%) in a 4 x 4 Latin square design. The remainder of the diet included chopped bermudagrass hay, dry-rolled corn, and protein/mineral supplement. Duodenal samples were collected for 4 d after 11-d diet adaptation periods. Data were analyzed with animal, period, oil source, fish oil, and 2-way interaction among oil source and fish oil quantity in the model. All interactions among oil source and fish oil inclusion were nonsignificant with the exception of trans-11 vaccenic acid (TVA) and trans-9 octadecenoic acid. Intake and duodenal flow of total long-chain fatty acids did not differ between treatments. Apparent ruminal DM digestibility was not altered by oil source or fish oil inclusion. Apparent ruminal digestion of fatty acids did not differ among oil sources but was increased (P = 0.03) with fish oil supplementation. Ruminal BH of oleic and linolenic acids was increased (P = 0.01) for diets containing supplemental canola oil compared with corn oil. Ruminal BH of linoleic acid was greater (P = 0.01) for diets containing supplemental corn oil compared with canola oil. Fish oil addition reduced (P = 0.01) oleic acid BH but did not alter (P > 0.26) linoleic or linolenic acid BH. Duodenal flow of palmitic acid was greater (P = 0.05) for steers supplemented with corn oil compared with canola oil. Fish oil inclusion in the diet increased (P = 0.01) flow of n-3 fatty acids (eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid), trans-10 octadecenoic acid, trans-12 octadecenoic acid, and cis-9, trans-11 CLA. Trans-9 octadecenoic acid and TVA flows to the duodenum were increased (P = 0.01) when fish oil was included in the canola oil-supplemented diet; however, no changes were observed when fish oil was included in the corn oil-supplemented diet (P of interaction = 0.06 and 0.08). Fish oil inclusion increased the outflow of n-3 fatty acids, trans-10 octadecenoic acid, and the majority of CLA isomers including cis-9, trans-11. These results suggest that fish oil addition alters ruminal formation of BH intermediates that is dependent on oil source supplemented in the diet.