The expression of rat GLUT5 is tightly regulated during development. Expression and activity are low throughout the suckling and weaning stages, but perfusion of the small intestinal lumen with fructose solutions during weaning precociously enhances GLUT5 activity and expression. Little is known, however, about the signal transduction pathways involved in the substrate-induced precocious GLUT5 development. We found that wortmannin and LY294002, inhibitors of phosphatidylinositol 3- kinase (PI3K) specifically inhibited the increase in fructose uptake rate and brush border GLUT5 protein abundance, but not GLUT5 mRNA abundance. Perfusion of EGF, an activator of PI3K, also resulted in a marked wortmannin-inhibitable increase in fructose uptake. Perfusion of fructose for 4 h increased cytosolic immunostaining of phosphatidylinositol-3,4,5-triphosphate (PIP3), the primary product of PI3K, mainly in the mid to upper villus regions where the brush border membrane also stained strongly with GLUT5. Perfusion of glucose for 4 h had little effect on fructose or glucose uptake, and PIP3 or GLUT5 staining. SH-5, an Akt inhibitor, prevented the increase in fructose uptake and GLUT5 protein induced by fructose solutions, and had no effect on glucose uptake. The PI3K/Akt signaling pathway may be involved in the synthesis and/or recruitment to the brush border of GLUT5 transporters by luminal fructose in the small intestine of weaning rats. Increases in fructose transport during the critical weaning period when rats are shifting to a new diet may be modulated by several signaling pathways whose cross- talk during development still needs to be elucidated.