Four Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square to investigate the effects of calcium salts of long-chain fatty acids (fat) and source of protein (fish meal or soybean meal) on ruminal fermentation, flow of nutrients to the small intestine, and animal performance. Cows were fed for ad libitum intake a diet of 30% alfalfa haylage, 20% corn silage, and 50% concentrate on a DM basis. Treatments, arranged in a 2 x 2 (fat x protein) factorial, were 1) soybean meal, no fat; 2) soybean meal, fat; 3) fish meal, no fat; and 4) fish meal, fat. Intake of DM was not affected by fat or protein source, but feeding fat decreased the amount of OM truly digested in the rumen. Starch intake was decreased, but flow of starch to the duodenum was not altered by feeding fat. Nonammonia N and microbial N flows to the duodenum were not affected by treatment comparisons. However, efficiency of microbial growth was increased by feeding fat, but not by source of protein. Passage of amino acids to the duodenum was not affected by source of protein, probably because fish meal contributed only 17% of the total dietary CP, and microbial N constituted about 50% of the NAN passing to the duodenum; this had an equalizing effect on the pattern and quantity of amino acids that passed to the duodenum. Feeding fat or different sources of protein did not alter milk production. Milk fat percentage was increased, and protein percentage was decreased when fat was fed, but yields of milk fat and protein were not different.