The extent and/or intermediates of ruminal biohydrogenation (BH) of fatty acids (FA) were investigated in vitro and in situ using a raw, pre-conditioned or extruded blend of linseed and wheat bran (70:30). The duration of in vitro incubations were 2, 4, 8, 16 and 24 h, with 5 replicates. In situ studies used 3 dry ruminally fistulated Holstein cows in a 3 × 3 Latin square design, with 3 weeks adaptation to the linseed form. The diet contained 20% (DM basis) of the linseed based blend. The duration of in situ incubations were 2, 4, 8, 16, 24 and 48 h. BH was much slower in situ than in vitro, resulting in a much lower effective disappearance of C18:2 and C18:3. Moreover, the in situ technique suggested that the technological pre-treatment of linseed did not affect C18:2 and C18:3 rate of BH, whereas reduced rates were observed in vitro. After 8 h of in vitro incubation and onwards, proportions of cis-9,trans-11C18:2 were the highest with extruded linseed. The BH of FA from linseed resulted in the appearance of great proportions of trans-10+11 to trans-16C18:1 intermediates. Extrusion increased the proportions of trans-10+11C18:1 both in vitro and in situ and proportions or trans-C18:1 were higher in situ than in vitro. Compared to previous in vivo results with the same material, the in situ method provided poor estimates of BH rates and intermediates