Conjugated linoleic acid (CLA), a naturally occurring anticarcinogen found in dairy products, is a byproduct of incomplete ruminal biohydrogenation of unsaturated fatty acids. Our objective was to determine the effect of nonstructural carbohydrate sources, addition of full fat extruded soybeans as a source of unsaturated fatty acids, and possible interactions on the milk fat content of CLA. Cows (n = 20) were assigned to a 4 x 4 Latin square involving two sources of nonstructural carbohydrate, high starch (corn) or high pectin (citrus pulp), with or without addition of extruded soybeans. Milk yield was not affected by nonstructural carbohydrate source, but milk production was increased by 7.8 to 10.5% with dietary additions of extruded soybeans. Milk fat content did not differ between treatments, but fatty acid composition was affected. Cows fed extruded soybean diets had reduced concentrations of C8 to C16 fatty acids and increased concentrations of octadecenoic acids. Diets with extruded soybeans also resulted in more than a doubling in milk fat concentration and yield of CLA. Nonstructural carbohydrate source had only minor effects on CLA, and there was no interaction with extruded soybeans. Milk fat content of trans-C18:1 and CLA were closely related (r2 = 0.77). However, variation among cows was about threefold for each of the diets and rank order of individual cows differed among diets. Overall, we demonstrated that diet modification can be used to alter CLA content of milk fat, but there was substantial individual cow variation for all diets.