Trans-10, cis-12-conjugated linoleic acid (CLA) is a potent bioactive fatty acids (FA) that causes milk fat depression in lactating animals. FA are transferred to milk directly through chylomicrons and indirectly by recycling through other tissues. The objective of this study was to characterise the kinetics of trans-10, cis-12 CLA transfer to plasma and milk after a single bolus infusion. Five multiparous mid-lactation cows received a single abomasal bolus infusion of an enriched CLA mixture providing 15 g of trans-10, cis-12 CLA and 15 g of cis-9, trans-11 CLA over a 30-min period. Plasma concentration of trans-10, cis-12 and cis-9, trans-11 CLA peaked 2 h post-bolus, reaching 0·29 and 0·38 % of total plasma FA, respectively, and returned to pre-bolus values at 72 h post-infusion. Milk trans-10, cis-12 CLA yield and concentration peaked 14 h post-bolus (0·25 g/h) and was not detectable in milk after 86 h. Total apparent transfer of trans-10, cis-12 CLA to milk was 41 %, with 73 % transferred to milk through the direct pool (chylomicrons) and the remaining 27 % transferred through the indirect pool (tissue recycling). Compartmental modelling revealed the existence of a transient unavailable pool of trans-10, cis-12 CLA in extravascular tissues represented primarily by the mammary gland, which slowly exchanges with an available pool for secretion in milk fat and transfer to milk. In conclusion, trans-10, cis-12 CLA is predominantly transferred to milk through the direct pathway; however, how this CLA isomer is processed within the mammary gland requires further investigation.