Using 6 continuous cultures of rumen microorganisms, we studied the effects of pH (low vs. high) and extruded linseed supplementation (10% of DM) in association with rapidly or slowly degraded starch sources (wheat vs. corn grains) 1) on fatty acid (FA) outflows and PUPA biohydrogenation (BH) processes 2) on FA microbial composition and its contribution to FA outflows, in two replicated periods of 10d (7d adaptation and 3d sampling). The control diet contained wheat. The buffer solutions infused in low or high pH cultures differed by the addition of 10 mL of 5 N H2SO4 or NaOH so as to allow fermentation medium pH fluctuations with diets and time after feeding. The fermentation pattern, daily amounts of hexoses fermented (HF), efficiency of microbial protein synthesis (EMPS), and specific production of gases (CH4 and H-2) were measured. FA compositions were determined in total effluents and bacterial pools isolated from effluents. Lowering pH (from 6.46 to 6.16 measured just before feeding) increased the VFA concentration in the control diet whereas it decreased it in all linseed supplemented diets. Lowering pH tended to decrease CH4 specific production as well as acetate and propionate proportions and to increase butyrate and valerate proportions but did not alter HF and EMPS. Linseed but not grain source increased pH by 0.08 and did not modify fermentative parameters. Apparent BH of C18:3 9c,12c,15c and C18:2 9c,12c decreased at low pH but increased with linseed supplementation. Corn associated to linseed increased 18:3 9c,12c,15c BH compared to wheat. Consequently, C18:3 9c,12c,15c outflows increased at low pH and with linseed, and were higher with linseed-wheat diet than linseed-corn diet. For all treatments, the proportions of C18:0 (% of C18-FA outflows) remained low associated with high levels of BH intermediates (C18:2 lit, 15c and C18:1 lit) suggesting that BH did not proceed to completion. Lowering pH decreased C16:1 9c and C18:1 11c bacteria contents and tended to increase anteiso-FA. Linseed supplementation increased C18:0, C18:1 9c, C18:1 9t, C18:2 9c,12c bacteria contents without modifying the C18:3 9c,12c,15c and decreased their odd-FA content and anteiso-FA in tendency. Compared to wheat, corn decreased branched-FA bacteria content, as well as even-saturated FA. Lowering pH decreased the bacterial FA outflow whereas linseed increased it. Both decreased the bacterial FA contribution to total FA outflow. Results indicate that pH and diets modified PUFA BH while differing in their effect on odd- and branched-chain FA bacteria content and outflows.