This investigation evaluated the adaptive response of the glycolytic enzymes, fructose-1-phosphate aldolase, fructose-1, 6-diphosphate aldolase, and pyruvate kinase, to dietary sugars throughout the small intestine. In addition, the effect of prior diet on this adaptive response and on the enzyme distribution pattern along the small intestine was studied. Rats were fed 40% glucose, 68% sucrose or carbohydrate-free diets for 6 days (baseline diet), followed by one of three isocaloric test diets (40% glucose, 68% sucrose or carbohydrate-free for 3 days. In other groups of tats isocaloric diets of 68% glucose, 68% fructose or 34% glucose + 34% fructose, fed for 4 days, were compared. Enzymes were assayed in the mucosa of the duodenum (D),and in 5 equal (by length) segments from the Ligament of Treitz to the ileocecal valve (J1, J2, J3, I1 and I2). Enzyme specific activities were significantly higher in the proximal (D-J1-J2) than distal segments (J3-I1-I2) on all diets (P smaller than 0.001). Enzyme activities after test diet periods were determined only by the test diet, and were independent of the baseline diet for all segments. The 68% carbohydrate diets increased enzyme activities significantly more (P smaller than 0.001) than the 40% glucose or carbohydrate free diets, in all segments. On the 40% glucose diet, activities were significantly higher (P smaller than 0.05) than on the carbohydrate free diet in D and J1, but not distally. The data suggest that there is an intrinsic gradient of enzyme activity from the proximal to the distal small intestine which persists despite dietary manipulation, and that all segments of the small bowel show adaptive increases to dietary sugars.