The effect of biotin deficiency and supplementation at pharmacological doses on the intestinal transport of the vitamin was examined in the rat using a brush-border membrane vesicle (BBMV) technique. Transport of biotin in both jejunal and ileal BBMV was significantly (P less than 0.05-0.01) higher in biotin-deficient rats compared with control (pair-fed) rats. This increase in biotin transport appeared to be specific because transport of D-glucose was similar in the two rat groups. The increase in biotin transport in the deficient rats appeared to be mediated through a marked increase (146-230%) in the Vmax of the biotin transport process (with minimal change in the apparent Km), suggesting an increase in the number of the transport carriers. In contrast, supplementation at pharmacological doses of biotin caused significant (P less than 0.05-0.01) and specific decrease (suppression) in biotin transport compared with (unsupplemented) controls. The suppression of biotin transport in the supplemented rats appeared to be mediated through a marked decrease (58%) in the Vmax of the biotin transport process (with minimal change in the apparent Km), suggesting a decrease in the number of the transport carriers. These results provide evidence that biotin transport in the intestine is regulated by the level of the vitamin in the diet (and/or body stores). Furthermore, the results demonstrate the ability of the small intestine to adapt to the challenge of deficiency of an essential nutrient, a capability that may be crucial for the survival of the animal.