GLUT8 is a newly identified member of the facilitative glucose transporter family, which characteristically exhibits high-affinity glucose transport activity. The expression of GLUT8 has been shown to depend on gonadotropin secretion in human testes and to be regulated by insulin in the blastocyst. To characterize GLUT8 and investigate its role in normal mammary gland function, we cloned and sequenced the full-length cDNA of bovine GLUT8. The 2073-base-pair cDNA sequence is predicted to encode a protein of 478 amino acids, with a molecular weight of approximately 51 kDa. The deduced amino acid sequence of bovine GLUT8 is 90%, 84%, 84% and 58% identical to human, mouse, rat and chicken GLUT8, and is 26%, 27% and 24% identical to bovine GLUT1, GLUT3 and GLUT4, respectively. Bovine GLUT8 retains the characteristic structural features of GLUT8 proteins previously identified from other species including membrane spanning helices, glucose transporter motifs, an N-linked glycosylation site on loop 9 and a putative dileucine internalization motif. The major in vitro transcription and translation product of bovine GLUT8 cDNA migrated at an apparent molecular weight of 38 kDa similar to the sizes reported for GLUT8 from other mammalian species. In the presence of canine microsomal membranes, the translation product increased to 40 kDa suggesting glycosylation. Transient transfection studies using a FLAG epitope tagged construct in COS-7 cells revealed that bovine GLUT8 is localized to the cytoplasm in non-stimulated conditions. A 2.1-kb GLUT8 mRNA transcript was detected at high levels in bovine testes, at moderate levels in lactating bovine mammary gland, lung, kidney, spleen, intestine and skeletal muscle, and at low levels in bovine liver. GLUT8 mRNA expression in bovine mammary gland increased about 10-fold (P<0.001) during late pregnancy and early lactation, similar to the pattern of change in GLUT1 mRNA and more dramatic than the increase seen in mouse mammary gland. These results suggest that GLUT8 expression may be regulated by lactogenic hormones and that GLUT8 may play a role in glucose uptake in the lactating mammary gland.