Mechanisms underlying milk fat conjugated linoleic acid (CLA) responses to supplements of fish oil were investigated using five lactating cows each fitted with a rumen cannula in a simple experiment consisting of two consecutive 14-day experimental periods. During the first period cows were offered 18 kg dry matter (DM) per day of a basal (B) diet formulated from grass silage and a cereal based-concentrate (0·6 : 0·4; forage : concentrate ratio, on a DM basis) followed by the same diet supplemented with 250 g fish oil per day (FO) in the second period. The flow of non-esterified fatty acids leaving the rumen was measured using the omasal sampling technique in combination with a triple indigestible marker method based on Li-Co-EDTA, Yb-acetate and Cr-mordanted straw. Fish oil decreased DM intake and milk yield, but had no effect on milk constituent content. Milk fat trans-11 C18:1, total trans-C18 : 1, cis-9 trans-11 CLA, total CLA, C18 : 2 (n-6) and total C18 : 2 content were increased in response to fish oil from 1·80, 4·51, 0·39, 0·56, 0·90 and 1·41 to 9·39, 14·39, 1·66, 1·85, 1·25 and 4·00 g/100 g total fatty acids, respectively. Increases in the cis-9, trans-11 isomer accounted for proportionately 0·89 of the CLA response to fish oil. Furthermore, fish oil decreased the flow of C18 : 0 (283 and 47 g/day for B and FO, respectively) and increased that of trans-C18 : 1 fatty acids entering the omasal canal (38 and 182 g/day). Omasal flows of trans-C18 : 1 acids with double bonds in positions from delta-4 to -15 inclusive were enhanced, but the effects were isomer dependent and primarily associated with an increase in trans-11 C18 : 1 leaving the rumen (17·1 and 121·1 g/day for B and FO, respectively). Fish oil had no effect on total (4·36 and 3·50 g/day) or cis-9, trans-11 CLA (2·86 and 2·08 g/day) entering the omasal canal. Flows of cis-9, trans-11 CLA were lower than the secretion of this isomer in milk. Comparison with the transfer of the trans-9, trans-11 isomer synthesized in the rumen suggested that proportionately 0·66 and 0·97 of cis-9, trans-11 CLA was derived from endogenous conversion of trans-11 C18 : 1 in the mammary gland for B and FO, respectively. It is concluded that fish oil enhances milk fat cis-9, trans-11 CLA content in response to increased supply of trans-11 C18:1 that arises from an inhibition of trans-C18 : 1 reduction in the rumen.