The influence of intestinal microbial bile salt deconjugation on absorption of fatty acids and α- and γ-tocopherol was investigated in a trial with Ross 208 broilers. Birds (n = 1600) were assigned to 4 dietary treatments: no supplementation or supplementation of antibiotics (salinomycin, 40 mg/kg feed and avilamycin, 10 mg/kg feed), and inclusion of either animal fat (10 g/100 g feed) or soybean oil (10 g/100 g feed) in the diet. At d 7, 14, 21, and 35 of age, the intestinal number of the bile salt hydrolase-active bacteria Clostridium perfringens, the concentration of conjugated and unconjugated bile salts, the ileal absorption of fatty acids and tocopherols, and the blood plasma concentrations of tocopherols were measured. All variables were significantly influenced by bird age. C. perfringens counts were lower and bile salt concentrations were greater in birds fed soybean oil. The supplementation of antibiotics reduced the numbers of C. perfringens in the small intestine and reduced the concentration of unconjugated bile salts. The ileal absorption of fatty acids and α-tocopherol, as well as the plasma concentration of α-tocopherol, was greater in birds fed antibiotics. The absorption and plasma concentration of γ-tocopherol were not influenced by antibiotics. Unlike γ-tocopherol, which is present solely as the free alcohol, the major proportion of dietary α-tocopherol is present as α-tocopheryl acetate, which requires a bile salt-dependent enzymatic hydrolysis before absorption. In conclusion, proper digestion of lipid-soluble compounds is highly dependent on an adequate concentration of bile salts in the small intestine to provide proper lipid emulsification and activation of lipolytic enzymes. J. Nutr. 134: 1487-1492, 2004.