Lactose, the major carbohydrate of human milk, is synthesized in the Golgi from glucose and UDP-galactose. The lactating mammary gland is unique in its requirement for the transport of glucose into Golgi. Glucose transporter-1 (GLUT1) is the only isoform of the glucose transporter family expressed in mammary gland. In most cells, GLUT1 is localized to the plasma membrane and is responsible for basal glucose uptake; in no other cell type is GLUT1 a Golgi resident. To test the hypothesis that GLUT1 is targeted to Golgi during lactation, the amount and subcellular distribution of GLUT1 were examined in mouse mammary gland at different developmental stages. Methods including immunohistochemistry, immunofluorescence, subcellular fractionation, density gradient centrifugation, and Western blotting yielded consistent results. In virgins, GLUT1 expression was limited to plasma membrane of epithelial cells. In late pregnant mice, GLUT1 expression was increased with targeting primarily to basolateral plasma membrane but also with some intracellular signal. During lactation, GLUT expression was further increased, and targeting to Golgi, demonstrated by colocalization with the 110-kD coatomer-associated protein beta-COP, predominated. Removal of pups 18 d after delivery resulted in retargeting of GLUT1 from Golgi to plasma membrane and a decline in total cellular GLUT1 within 3 h. In mice undergoing natural weaning, GLUT1 expression declined. Changes in the amount and targeting of GLUT1 during mammary gland development are consistent with a key role for GLUT1 in supplying substrate for lactose synthesis and milk production.