Diet is a relatively simple way to modify milk fat yield and composition in dairy cows as the end-products of digestion are precursors or inhibitors of milk fat synthesis. The individual effects of these end-products are well-known, but it is still not known whether these nutrients have an additive effect or an interaction effect on milk fat secretion. Thus our objective was to investigate the effects of two of these nutrients on milk fat secretion, trans-10, cis-12 conjugated linoleic acid (CLA) and propionic acid (C3) supplied alone or together, under the same experimental conditions. Four Holstein dairy cows were used in a 4×4 Latin square design with 14-d periods. Treatments were control, CLA (duodenal infusion of 1·85 g/d of trans-10, cis-12 CLA), C3 (ruminal infusion of 500 g/d of C3) and CLA+C3 (duodenal infusion of 1·85 g/d of trans-10, cis-12 CLA plus ruminal infusion 500 g/d of C3). Infusions of trans-10, cis-12 CLA reduced milk fat content and yield by 18% whereas C3 infusions had no significant effect on milk fat secretion. Trans-10, cis-12 CLA decreased the yields of all milk fatty acids (FA). This reduction was proportionally greater for FA synthesized de novo than for preformed long-chain FA. Infusions of C3 decreased the yields and percentages of 4:0 and 18:0 and increased the yields and percentages of all odd-chain FA. Interactions between trans-10, cis-12 CLA and C3 infusions on milk fat content, yield and FA composition were never significant. Overall, this study showed that trans-10, cis-12 CLA has different and greater effects on milk fat secretion than C3. Moreover, under our experimental conditions, their effects on milk FA yields, which reflect their effects on mammary lipogenesis, were additive, whatever their individual effect.