The effect of niacin supplementation on digestion was investigated using four cows in a crossover design. After peak lactation, cows received a diet of corn silage (55%) and concentrates (45%), including a formaldehyde-treated mixture of soybean meal, rapeseed meal, and urea. This diet was either supplemented with niacin (6 g/d) or not supplemented. Total tract apparent digestibility of OM and fiber and ruminal digestibility of OM were not modified by treatment. Niacin supplementation enhanced the theoretical degradability of ruminal DM in situ (44.7% vs. 40.6%) and concentration of protozoa in ruminal fluid (459 vs. 311 x 10(3)/ml), especially Ophryoscolecidae. The percentage of butyrate in the VFA mixture of ruminal fluid was increased by niacin supply, but acetate and propionate percentages and total VFA concentration did not vary. Ruminal digesta, ruminal pools, and ruminal kinetics were not affected by treatment. Duodenal flow of nonmicrobial N tended to increase with niacin supplementation. Duodenal flow of microbial N did not vary, as measured using a microbial sample, for fluid-associated bacteria or for a mixture of fluid-associated and particle-associated bacteria. These results are discussed in relation to the characteristics of the diet.