The factors driving the progress of diet-induced milk fat depression (MFD) were studied by combining data from the flow of specific ruminal biohydrogenation (BH) intermediates of fatty acids (FA) at the omasum and the proportions of FA in milk fat over time. Four Nordic Red cows in mid-lactation were used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of diets and 35-d periods. Diets were based on grass silage: low (65:35, forage:concentrate, FC) or high (35:65) concentrate diets supplemented with 0 g (diets L and H, respectively) or 50 g/kg dry matter of sunflower oil (SO; LSO and HSO, respectively). Data were analyzed using the MIXED procedure of SAS with fixed effects of period, FC, SO and FC × SO interaction. HSO lowered (FC × SO P < 0.01) milk fat synthesis up to 31.9% relative to other diets. MFD was associated with shift in ruminal BH toward the trans-10 pathway (trans, t; cis, c) and increases in t10 containing intermediates in milk. However, the flow of t10,c12 conjugated linoleic acid (CLA) at the omasum was greater (P < 0.01) on both H and HSO compared with L and LSO indicating that t10,c12 CLA was not the only factor causing MFD. Contrary to this, MFD on the HSO was accompanied by an increase in t10,c12 CLA in milk compared with other diets (FC × SO P < 0.05). HSO resulted in the formation of t10 18:1 and t9,c11 CLA in the rumen relative to other diets, causing increased proportions of these intermediates also in milk (FC × SO P < 0.05), which support the previous evidence that t10 18:1 and t9,c11 CLA have a role in the regulation of milk fat synthesis. BH intermediate t10,c15 18:2 was found in omasal digesta and milk on HSO providing more support to the existence of alternative t10 pathway of 18:3n-3 metabolism in the rumen that is pronounced in cows fed diets causing MFD. In conclusion, ruminal t10,c12 CLA formation was not accompanied by MFD suggesting that other BH intermediates (t10 18:1, t9,c11 CLA, t10,c15 18:2) or additional mechanisms have a role in the regulation of fat synthesis in the bovine mammary gland.