The additional mass associated with 13C in metabolic tracers may interfere with their metabolism. The comparative isomerization and biohydrogenation of oleic, [1-(13)C]oleic, and [U-13C]oleic acids by mixed ruminal microbes was used to evaluate this effect. The percent of stearic, cis-14 and -15, and trans-9 to -16 18:1 originating from oleic acid was decreased for [U-(13)C]oleic acid compared with [1-(13)C]oleic acid. Conversely, microbial utilization of [U-(13)C]oleic acid resulted in more of the 13C label in cis-9 18:1 compared with [1-(13)C]oleic acid (53.7 vs. 40.1%). The isomerization and biohydrogenation of oleic acid by ruminal microbes is affected by the mass of the labeled tracer.