Lactating cows with cannulae in the rumen and proximal duodenum were used in two 4 X 4 Latin square experiments to evaluate biohydrogenation, flow rates, and digestibility of dietary fatty acids in the gastrointestinal tract. In the first experiment, four cows were fed diets with 0% supplemental fat, 3% tallow, 3% partially hydrogenated tallow, or 3% tallow coated with casein and corn syrup solids. Fatty acid intake and flow to the duodenum increased with fat supplementation. Total tract apparent fatty acid digestibility was reduced by partially hydrogenated tallow. Apparent digestibility of C_18:0 in the small intestine quadradically decreased (R²=.86) as C_18:0 flow to the intestine increased. Fat supplementation increased milk production and decreased milk protein percent but did not affect milk fat percent or 3.5% FCM. Fat supplementation increased C_18:0 and C_18:1 and lowered C_12:0 and C_14:0 concentrations in milk fat. In the second experiment, four lactating Holstein cows were fed diets with 0% supplemental fat, 3% partially hydrogenated fatty acids, 1.5% fish oil plus 1.5% stearic acid, or 1.5% soybean oil plus 1.5% partially hydrogenated soybean oil. Fish oil decreased DM intake. Fish oil and soybean oil reduced biohydrogenation of C_18:1 + C_18:2 + C_18:3 in the rumen from 65% (control) to 28% and 55%, respectively, and increased trans C_18:1 flow from the rumen 4-fold. Milk fat percent was decreased by fish oil and soybean oil. Milk fat percent across treatments linearly decreased with amount (g/d) of trans C_18:1 flowing to the duodenum (R²=.92) and percent trans C_18:1 in milk fat (R²=.94). / Master of Science