Abstract: Biosynthesis of milk conjugated linoleic acid (CLA), a component of milk fat with demonstrated health benefits, requires a dietary source of PUFA. Even with PUFA supplementation, milk CLA is highly variable. Therefore, this study was aimed at identifying factors responsible for the variations in rumen CLA precursors and milk CLA. Study 1 evaluated the efficiency of CLA production by grazing cows compared to those fed grass silage or fresh grass. Grazing cows were more efficient than those fed grass silage or fresh grass in milk CLA production. About 75% of the variability in milk CLA was related to the differences in PUFA (l8:2n-6 + 18:3n-3) intake and the remainder was related to factors regulating the extent of PUFA biohydrogenation in the rumen. This study demonstrated that PUFA intake is important but it is not the only factor responsible for the observed variation in milk CLA production. Study 2 evaluated the effect of diets differing in rate of starch degradation on rumen PUFA biohydrogenation and milk CLA. Concentrations of ruminal t11-18:1 and milk CLA were greater for barley-based diets than corn-based diets and were not different between rolling and grinding, indicating that factors inherent in the source of starch were responsible for the observed differences and these factors could not be modified by rolling or grinding the grain. Study 3 examined the effect of stage of lactation on persistency of milk t10-18:1, t11-18:1 and CLA for control and test (supplemented with PUFA and monensin) diets from calving to 270 days in milk. Milk concentrations of t11-18:1 and RA remained similar across the lactation length and were greater for the test diet compared to the control. Changes in milk t10-18:1 concentration during lactation appeared to reflect an effect of the degree of rumen fermentation on PUFA biohydrogenating bacteria. Although PUFA intake is important for milk CLA production, only those diets that give rise to increased ruminal t11-18:1 result in greater milk CLA. Concentrations of rumen t11-18:1 is influenced by the amount of PUFA consumed, degree of shift to t10-18:1 and the extent of PUFA biohydrogenation in the rumen.