The objectives of the present study were to determine the effect of inoculating Propionibacterium freudenreichii subsp. shermanii ATCC 8262 (1 x 10(9) colony-forming units per vial) in a barley silage-based diet supplemented with flaxseed oil or rapeseed oil (60 g/kg DM), on in vitro proportions and yield of volatile fatty acids, methane production and fatty acid (FA) biohydrogenation. Total volatile fatty acid production (mM) and proportions of individual FAs were not affected (P >= 0.10) by P. freudenreichii. Similarly, propionibacteria had little impact onFAbiohydrogenation, resulting only in an increased accumulation (P<0.01) of C18: 1 cis-15 (g/kg total FA) at 6 h of incubation, compared with the control (CON). Compared with the CON, an increased (P < 0.01) accumulation of vaccenic acid was observed at 48 h in all oil-containing treatments, regardless of the oil type. Similarly, the apparent biohydrogenation of flaxseed oil resulted in an increased (P <= 0.04) accumulation of conjugated linoleic acid cis-9, trans-11, compared with all other treatments. Additionally, flaxseed oil produced a greater (P <= 0.01) accumulation of beneficial biohydrogenation intermediates (C18: 2 trans-11, cis-15; C18: 1 cis-15 and vaccenic acid), reflecting its ability to produce a more desirable FA profile than that of rapeseed oil or CON. The inability of P. freudenreichii subsp. shermaniiATCC8262 to alter ruminal fermentation in a manner that lowered methane production, along with only minor effects on FA profiles through biohydrogenation, suggests that the biological activity of this strain was not realised under in vitro batch-culture conditions.