It has been previously established that trans-10, cis-12 conjugated linoleic acid plays an important role in milk fat depression (MFD). However, in many situations of dietary induced MFD, the reduction in milk fat synthesis is much greater than what would be predicted based on the milk fat concentration of trans-10, cis-12 18:2. These observations suggest that other biohydrogenation intermediates could be implicated in MFD. The objective of this study was to evaluate the effects on milk fat synthesis of an intravenous administration of 2 conjugated diene 18:3 isomers (cis-9, trans-11, cis-15 and cis-9, trans-13, cis-15 18:3), which are intermediates in ruminal biohydrogenation of alpha-linolenic acid. Three multiparous Holstein dairy cows (days in milk = 189 +/- 37 d; body weight = 640 +/- 69 kg; mean +/- standard deviation), fitted with indwelling jugular catheters, were randomly assigned to a 3 x 3 Latin square design. For the first 5 d of each period, cows were infused intravenously with a 15% lipid emulsion providing 1) cis-9, trans-11, cis-15 18:3 + cis-9, trans-13, cis-15 18:3 + trans-10, cis-12 18:2 (CD18:3 + CLA); 2) cis-9, cis-12, cis-15 18:3 + cis-9, cis-12 18:2 as a control (ALA + LA); or 3) cis-9, cis-12, cis-15 18:3 + trans-10, cis-12 18:2, as a positive control (ALA + CLA). Milk production was recorded, and milk was sampled daily at each milking for analyses of fat, protein, lactose, milk urea nitrogen, and somatic cell count. Dry matter intake, milk yield, and milk protein were not affected by treatment. Over the experimental period, milk fat content was decreased by 7% for cows that received either ALA + CLA or CD18:3 + CLA compared with ALA + LA. The temporal pattern of milk fat content showed a linear decrease during the infusion period for ALA + CLA and CD18:3 + CLA treatment groups. The transfer efficiencies of conjugated diene 18:3 isomers into milk fat averaged 39 and 32% for cis-9, trans-11, cis-15 18:3 and cis-9, trans-13, cis-15 18:3, respectively. The CD18:3 + CLA treatment had no effect on milk fat concentration beyond that attributable to its trans-10, cis-12 18:2 content. In conclusion, results from the current study offered no support for a role of either cis-9, trans-11, cis-15 18:3 or cis-9, trans-13, cis-15 in MFD.