Synthesis from glucose of lactose and lipid within the lactating mammary gland determines the volume and caloric content of milk. Glucose must be transported not only across the basal plasma membrane of mammary epithelia(ME), but also into the Golgi, the site of lactose synthesis. My purpose was to determine changes in the subcellular localization of glucose transporters in ME cells in response to lactogenic hormones. Mouse CIT3 ME cells (kindly supplied by Dr. P. Neville, Univ. of Colorado), derived from Comma-1-D cells, are maintained in the presence of insulin and EGF (growth medium, GM) and differentiate in the presence of prolactin and dexamethasone (secretion medium, SM). Differentiation of CIT3 cells causes an increase in total cellular GLUT1 glucose transporters but a decrease in the proportion of GLUT1 at the cell surface. To test the hypothesis that GLUT1 is sequestered in the Golgi under these conditions, the subcellular localization of GLUT1 and of BODIPY-TR, a Golgi marker, was defined by dual-label immunocytochemistry. This demonstrated the localization of GLUT1 in Golgi. To study possible polarized targeting of GLUT1 to plasma membrane domains, cells were grown on tissue culture inserts. Basal membrane glucose transport activity was not significantly different in SM compared to GM, but apical membrane glucose transport activity fell 86%, so that the ratio of basal to apical glucose transport activity changed from 3:2 in GM to 9:1 in SM. Thus, differentiation of ME cells alters the subcellular localization of ME glucose transport activity such that it is targeted to Golgi and basal membrane, helping to meet the metabolic demands of lactation.