Mouse GLUT5 cDNA and a 7.7-kb genomic fragment have been isolated and characterized. The cDNA sequence suggests mouse GLUT5 is composed of 501 amino acids, and has 69-88% amino acid identity with human, rat, and rabbit GLUT5. Expression of mouse GLUT5 cRNA in Xenopus laevis oocytes showed that GLUT5 mediated fructose transport, with a K(t) of 13 mM. Northern blot studies detected GLUT5 mRNA expression in mouse small intestine, kidney, and testis, with transcript sizes of approximately 2.1, 2.1, and 2.8 kb, respectively. 5'Rapid Amplification of cDNA Ends (5'RACE) determined that the differences in transcript sizes occurred because GLUT5 possessed alternative transcriptional initiation sites in somatic and germ cells. In agreement with studies in rats and rabbits, mouse small intestinal GLUT5 mRNA expression levels were increased following exposure to a 65% fructose-enriched diet. In addition, developmental studies showed a significant increase in GLUT5 mRNA expression levels in adult mouse testis when compared to prepubertal mouse testis. To begin to identify the cis-acting domains responsible for GLUT5 expression characteristics, a 7.7-kb GLUT5 genomic fragment was isolated from a mouse lambda fix11 library and sequenced. The clone contained exons 1-4 and 5' flanking regions. Moreover, caudal homeobox gene (CdxA), upstream stimulatory factor (USF), and sex-determining region of Y (SRY) binding sites were identified in the 5' flanking region that may be responsible for GLUT5's expression characteristics: tissue distribution, sensitivity to dietary fructose in the small intestine, and developmental expression in the testis.