Biohydrogenation (BH) of unsaturated fatty acids (FA) has been extensively studied in vitro but BH rates and intermediates formed in vitro may not parallel BH pathways in vivo. The objective was to develop an in vivo method to determine the rate of α-linolenic acid (18:3 n-3) BH and identify intermediates formed. Eleven rumen cannulated high-producing Holstein cows [40 ± 6 kg milk/d (Mean ± SD)] were fed at a rate of 6%/h of expected total DMI a diet balanced to 29% NDF and 5.9% EE (1.5% soybean oil). A single bolus consisting of 200 g of flaxseed oil (53% 18:3) and 15 g of tridecanoic acid (13:0) was mixed with rumen contents and rumen digesta was collected at −1, 0.1, 0.5, 1, 2, 3, 4, 6 and 8 h relative to the bolus. Samples were immediately placed in dry ice, stored at −20°C, freeze-dried, methylated and analyzed by gas chromatography. Data were first analyzed using PROC Mixed with repeated measures for time point comparison. Second, the disappearance of 13:0 and 18:3 was fit to a single exponential decay model using the nonlinear procedure of JMP Pro. The bolus increased total fat in the rumen from 4.3 to 6.0% and enriched 13:0 from 0.04 to 2.2% of FA and 18:3 from 2.0 to 11.3% of FA. The fractional rate of disappearance of 13:0 was 0.4%/min (r² = 0.98) and of 18:3 was 2.5%/min (r² = 0.99), with 18:3 reaching pre-bolus concentration within 4 h. Assuming that 13:0 disappeared only by passage, 18:3 disappeared by passage and biohydrogenation, and the rate of passage of 13:0 and 18:3 are the same, the extent of bolused 18:3 BH was 85%. The concentration of cis-9,trans-11,cis-15 18:3 peaked at 1.2% of FA at 1 h (8-fold increase), trans-11, cis-15 18:2 peaked at 3.9% of FA at 2 h (13-fold increase), and trans-11 18:1 peaked at 6.6% FA at 3 h (43% increase). In conclusion, the in vivo method resulted in the expected extent of biohydrogenation and biohydrogenation intermediates, but the rate of ruminal biohydrogenation of 18:3 was much higher than that commonly observed in vitro. The method developed provides an in vivo assay of ruminal biohydrogenation for use in future experiments.