Understanding ruminal biohydrogenation of linoleic and linolenic acid is important in relation to physiological responses in the animal and the fatty acid profile of ruminant meat and milk. Alterations in ruminal biohydrogenation pathways leading to an increased formation of trans-10-18:1 are known to occur with high-concentrate diets and marine supplements. We hypothesized that accumulation of trans-11-18:1 is a prerequisite for trans-10-18:1 production. To evaluate this hypothesis, a batch-culture method, using rumen fluid from wethers, was used which consisted of two periods. Period 1 (10 h) was used to induce changes in trans-11-18:1 accumulation using a 2 × 2 factorial design, with 18:2n-6 (0 vs 6.40 mg) and 22:6n-3 (0 vs 2.50 mg) replicated with three substrates (starch, glucose or cellobiose). As planned, the addition of 18:2n-6 in combination with 22:6n-3 resulted in greater accumulation of trans-11-18:1 than did the other treatments (2.73 ± 0.125 vs 0.37 ± 0.157 mg/flask). After P1, 18:2n-6 (3.20 mg) was added to all flasks and after 14 h of incubation, formation of trans-10-18:1 and trans-11-18:1 was evaluated. The apparent production of both trans-10-18:1 (0.057 vs 0.812 mg/flask) and trans-11-18:1 (–0.013 vs 1.100 mg/flask) for cultures receiving 22:6n-3 in P1 was greater independent of 18:2n-6 addition in P1 (P > 0.10). This lack of a significant interaction suggests that trans-11-18:1 accumulation was not a major factor explaining trans-10-18:1 production under the studied conditions.