The effect of acute changes in insulin concentrations in vivo on the absorption, transport and metabolism of glucose by rat small intestine in vitro was investigated. Within 2 min of the injection of normal anaesthetized rats with anti-insulin serum, lactate production and glucose metabolism were respectively diminished to 28% and 21% of normal and the conversion of glucose into lactate became quantitative. These changes correlated with the inhibition of two mucosal enzymes, namely the insulin-sensitive enzyme pyruvate dehydrogenase, and phosphofructokinase, which was shown by cross-over measurements to be the rate-limiting enzyme of glycolysis in mucosa. The proportion of glucose translocated unchanged from the luminal perfusate to the serosal medium was simultaneously increased from 45% to 80%. All the changes produced by insulin deficiency were completely reversed with 2 min when antiserum was neutralized by injection of insulin in vivo. The absorption and transport of 3-O-methylglucose were unaffected by insulin. It is concluded that glucose metabolism in rat small intestine is subject to short-term regulation by insulin in vivo and that glucose absorption and transport are regulated indirectly in response to changes in metabolism. Moreover, transport and metabolism compensate in such a way as to deliver the maximal 'effective' amount of glucose to the blood, whether as glucose itself or as lactate for hepatic gluconeogenesis.