The initial distribution and absorption of biotin in chickens was studied in vivo from either changes in the composition of intestinal contents or analysis of blood draining the gut. The progressive decline in biotin concentration and flow along the small intestine demonstrated absorption throughout this organ. Quantitatively the duodenum was the most significant site of biotin absorption when crystalline biotin was included in the diet and when dietary ingredients contained highly available biotin. In contrast, when diets of low biotin availability were given the distal segments of the small intestine contributed a relatively greater amount to biotin absorption. During passage of digesta from the ileum to the caecum there was a tenfold increase in biotin concentration. About half the amount was of ileal origin with the remainder presumably resulting from microbial synthesis. About 20% of caecal biotin was in a ‘free’ form but the degree of absorption from the caeca was not determined. The amount of biotin disappearing from the gut of birds given maize-based diets was similar to the quantity appearing in portal blood. Biotin absorption was not detected from wheat-based diets using arteriovenous differences.