To identify the mechanisms whereby norepinephrine induces glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. Proliferating brown adipocytes had high levels of GLUT1 mRNA and low levels of GLUT4 mRNA. The ratio of GLUT4/GLUT1 mRNA expression increased during differentiation and mature brown adipocytes had high levels of GLUT4 mRNA. The endogenous adrenergic neurotransmitter norepinephrine induced a potent increase in GLUT1 mRNA and a decrease of GLUT4 mRNA in mature brown adipocytes. The norepinephrine effect was mimicked by isoprenaline and CL 316243 and was thus mediated by ₃-adrenergic receptors. The cAMP analog 8-bromoadenosine-cAMP partly mimicked the response on GLUT1 mRNA increase and fully mimicked the GLUT4 mRNA decrease. We found no involvement of ₁ or ₂-adrenergic receptors on GLUT1 or GLUT4 mRNA transcription. Norepinephrine treatment led to a large increase of GLUT1 protein amount in brown adipocytes as visualized with immunocytochemical staining and subcellular fractionation. A large part of the newly synthesized GLUT1 was found in the plasma membrane. The potent transcriptional inhibitor actinomycin D fully abolished this increase of GLUT1 protein at all time points examined. Norepinephrine treatment shifted GLUT4 from the plasma membrane to an intracellular vesicular compartment. Norepinephrine increased 2-deoxy-D-glucose uptake 2-fold at an early time point (1 h) and 4-fold at later time point (5 h). Addition of actinomycin D did not block the early phase but blocked a large part of the later phase of 2-deoxy-D-glucose uptake. These results imply that adrenergic stimulation through ₃-adrenergic receptors induces glucose uptake in brown adipocytes via two mechanisms: 1) a mechanism not dependent on GLUT1 and GLUT4 translocation, 2) a mechanism which is dependent on de novo synthesis of GLUT1 protein and increase of GLUT1 protein at the plasma membrane.