In rodents, a number of intestinal digestive and absorptive processes demonstrate a diurnal pattern of activity. To investigate if the jejunal hexose transporters are regulated in such a diurnal fashion, the levels for the glucose and fructose transporter mRNA and proteins were determined at 6-h intervals over a 24-h control fed period. SGLT-1, GLUT-2, and GLUT-5 mRNA levels increased between two- and eightfold before the onset of peak feeding. GLUT-5 protein levels also varied in a diurnal fashion but were out of phase with the observed changes in GLUT-5 mRNA levels. In contrast, GLUT-2 protein levels remained relatively constant during the control fed 24-h period. The effect of dietary manipulations on the observed diurnal variation was also investigated. After only 3 h of feeding a 60% fructose-enriched diet, the levels of GLUT-5 mRNA and protein were significantly elevated. GLUT-5 mRNA and protein levels remained elevated relative to the level of control diet-fed animals over the ensuing 24 h and during the 7th day of fructose feeding. Exposure to elevated levels of fructose had no significant effect on the diurnal pattern of GLUT-2 and SGLT-1 mRNA. In contrast, GLUT-2 protein was rapidly downregulated during the length of the fructose feeding study. In conclusion, the data demonstrate a normal daily variation in the level of hexose transporter expression that can be rapidly modulated by diet.