The present experiment was designed to estimate the quantitative contribution of rumen protozoa to the total N, conjugated linoleic acid (CLA) and vaccenic acid (VA; trans-11-18 : 1) flow to the duodenum of steers fed two silage diets: control silage (CS) and silage high in water-soluble carbohydrates (HS). Protozoal duodenal flows were estimated using a real-time PCR assay to quantify the genes encoding protozoal 18S ribosomal RNA. Denaturing gradient gel electrophoresis was used to confirm that the rumen protozoa populations were similar to the protozoal population flowing to the duodenum. Estimated duodenal flow of protozoal N was 14.2 and 18.2 g/d (P > 0.05) for animals fed the CS and HS diets respectively. Protozoal flow thus represented between 12 and 15 % of the total N duodenal flow. In terms of fatty acid flow, protozoa accounted for between 30 and 43 % of the CLA and 40 % of the VA reaching the duodenum. The contribution of protozoa to 16 : 0 and 18 : 0 flows to the duodenum was less than 20 and 10 %, respectively. These results show that the fatty acids within protozoa make up a significant proportion of the CLA and VA reaching the duodenum of ruminants.