Specific activities of the glycolytic enzymes fructose-1-phosphate aldolase, fructose- 1,6-diphosphate aldolase, and pyruvate kinase, are higher in rat duodenum and jejunum than in ileum. Whether this gradient reflects the failure of dietary sugars to reach the ileum in high concentrations is unknown. Rats were first fed a carbohydrate-free diet for 3 days, which virtually removed the proximal-distal gradient of enzyme specific activities. Twenty percent glucose was then perfused directly into either the duodenum or the ileum for 3 days. Both proximal and distal glucose perfusion restored the normal gradient of all three enzymes. Ileal pyruvate kinase was also increased by ileal glucose perfusion, but ileal aldolases were no higher after distal perfusion than after duodenal perfusion. The low ileal aldolase levels normally found in fed rats therefore are an intrinsic property of distal intestine and are not due to failure of dietary sugar to reach this portion of the gut. Furthermore, adaptation of duodenal and jejunal glycolytic enzymes to ileal glucose perfusion suggests a humoral and/or neural mechanism rather than a direct local luminal effect of the sugar itself.