Tumor necrosis factor (TNF) is a potent inducer of insulin resistance and increased TNF expression is associated with impaired glucose disposal. While insulin is the primary regulator of glucose transport in adipose, endothelin-1, a vasoconstrictor peptide which signals through the heterotrimeric G-proteins G_q/11, potently stimulates glucose uptake in 3T3-L1 adipocytes by a mechanism independent of phosphatidylinositol-3 kinase (PI-3 kinase). Here we report that exposure of 3T3-L1 adipocytes to TNF for 48 h dose-dependently decreased endothelin-1-stimulated glucose uptake and translocation of GLUT4 to the plasma membrane. TNF exposure had no effect on endothelin-1 receptor number at the cell surface. In contrast, TNF treatment reduced the quantity of G_q/11 and PYK2 (proline-rich tyrosine kinase 2) and decreased endothelin-1-stimulated PYK2-Tyr⁴⁰² tyrosine phosphorylation. Taken together, these results suggest that TNF-induced desensitization of endothelin-1 stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes is due, at least in part, to a decreased expression of G_q/11, leading to a suppression in tyrosine phosphorylation of PYK2.