Aflatoxin B1 (AFB1) transfer across the gastrointestinal tract was studied in rats. The rate of biliary secretion of 3H was higher when [3H]AFB1 was injected into the small intestine than when injected into the stomach. When various sites of the small intestine were perfused with the medium containing [3H]AFB1, the highest rate of disappearance of 3H from the medium was noted in the duodenum. Also the rate of biliary secretion of 3H tended to be higher when the duodenum was perfused than when the other sites were perfused. These results suggest that AFB1 is absorbed mainly from the small intestine, most efficiently from the duodenum. Uptake of AFB1 by the everted intestine in vitro was slightly greater in the jejunum than the other sites, suggesting that the cause of the differences in the intestinal absorption among various sites may reside in the transfer process of AFB1 from the epithelial cell layer to vascular circulation. Comparison of the AFB1 appearance in the mesenteric venous plasma and lymph showed that AFB1 is absorbed almost exclusively in the vein. Distribution of 3H in the mesenteric plasma on thin-layer chromatography revealed that metabolic degradation of AFB1 takes place in the duodenal and jejunal tissues during the course of AFB1 absorption. Examination of the appearance of AFB1 or 3H in the intestinal perfusate after iv injection of of AFB1 or [3H]AFB1 suggested that AFB1 and its metabolites can transfer from the blood to the intestinal lumen. The rate of the AFB1 absorption from various sites of the intestine changed with age and reproductive stage, indicating that the AFB1 transfer across the intestinal wall is under the influence of the growth and reproductive endocrine condition.