The adaptation of d-fructose transport in rat jejunum to experimental diabetes has been studied. In vivo and in vitro perfusions of intact jejunum with d-fructose revealed the appearance of a phloretin-sensitive transporter in the brush-border membrane of streptozotocin-diabetic rats which was not detectable in normal rats. The nature of the transporters involved was investigated by Western blotting and by d-fructose transport studies using highly purified brush-border and basolateral membrane vesicles. GLUT5, the major transporter in the brush-border membrane of normal rats, was not inhibited by d-glucose or phloretin. In contrast, GLUT2, the major transporter in the basolateral membrane of normal rats, was strongly inhibited by both D-glucose and phloretin. In brush-border membrane vesicles from diabetic rats, GLUT5 levels were significantly enhanced; moreover the presence of GLUT2 was readily detectable and increased markedly as diabetes progressed. The differences in stereospecificity between GLUT2 and GLUT5 were used to show that both transporters contributed to the overall enhancement of d-fructose transport measured in brush-border membrane vesicles and in vitro isolated loops from diabetic rats. However, overall d-fructose uptake in vivo was diminished. The underlying mechanisms and functional consequences are discussed.