The lipid quality of ruminant products is largely determined by the extent of rumen microbial biohydrogenation (BH) of polyunsaturated fatty acids (FAs) and the substances formed thereby. In vitro batch incubations with mixed rumen bacteria were tracked over 24 h to characterize the profiles and kinetics of the BH products from three lipid sources: pure linoleic acid (c9,c12-18:2), pure linolenic acid (c9,c12,c15-18:3) and linseed oil (mainly c9,c12,c15-18:3 in triacylglycerols). After 24 h of incubation biohydrogenation was more complete for c9,c12-18:2, which gave mainly stearic acid (18:0), than for c9,c12,c15-18:3, which yielded mainly trans-18:1 FAs. This suggests inhibition of the final BH step (18:1 to 18:0). Incubations of c9,c12-18:2 resulted in high levels of carbon 10- and 12-desaturated 18:1, t10,c12- and c9,t11-CLAs. Incubations of c9,c12,c15-18:3 resulted in high levels of t11-18: 1, carbon 13- and 15-desaturated 18:1 as well as t11,c15-18:2 and 11,13-CLAs. A comparative study of linolenic acid and linseed oil kinetics revealed that the BH process was not significantly slowed by the esterification of polyunsaturated FAs, but may have been limited by the isomerization step in which the cis12 double bond goes to the trans1 I position. The disappearance rates of c9,c12-18:2 and c9,c12,c15-18:3 ranged from 23.6 to 44.6%/h. The wide variety of BH intermediates found here underlines the large number of possible BH pathways. These data help provide a basis for dynamic approaches to BH processes.