Uncoupling protein-2 (UCP-2) is a mitochondrial protein expressed in adipocytes and has recently been involved in the control of energy dissipation. Because obesity is characterized by an imbalance between energy intake and expenditure and by an enhanced adipocyte-derived secretion of tumor necrosis factor- (TNF-), we asked whether TNF- could directly influence UCP-2 expression in adipocytes. Experiments performed in differentiated 3T3F442A preadipocytes showed that TNF- (10 ng/ml) induced a reduction of UCP-2 trancripts, assessed by Northern blot analysis. A significant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF- stimulation of the cells. The characterization of the mechanisms responsible for the TNF- effect on UCP-2 expression demonstrates an involvement of the TNF--induced inducible (i) nitric oxide synthase (NOS) expression. Cell treatment with the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 1 mmol/l) significantly diminished the TNF--mediated sustained downregulation of UCP-2 expression, whereas cell treatment with a nitric oxide (NO) donor (10³ mol/l S-nitroso-L-glutathione) mimicked the TNF- effect on UCP-2 expression. Moreover, Western blot analysis clearly showed that TNF- alone induces the expression of iNOS after 12-24 h treatment of differentiated 3T3F442A cells. These experiments demonstrate that TNF- directly downregulates UCP-2 expression via NO-dependent pathways that involve the induction of iNOS expression.