It has been reported that tumor necrosis factor-alpha (TNF-alpha) inhibits insulin action in adipocytes and plays an important role as mediator of insulin resistance in non-insulin-dependent diabetes. The effect of this cytokine on insulin action in muscle, which is responsible for 80% of the glucose disposal in the body, has not been studied. Therefore, we examined the effect of TNF-alpha on basal and insulin-mediated transport of 2-deoxy[3H]-glucose in L6 rat muscle cells. TNF-alpha treatment for 5 days up to a concentration of 20 ng/mL or 8 days at 10 ng/mL did not inhibit the insulin-stimulated increase in deoxyglucose transport in L6 cells. However, there was a significant increase in basal transport in TNF-alpha- treated cells. Comparative experiments with 3T3-L1 adipocytes showed that in cells cultured with insulin, TNF-alpha decreased basal transport but the insulin-stimulated increase was unaffected. In cells cultured without insulin, basal transport was slightly increased and the insulin-stimulated increase in transport was decreased by approximately 60% but the cell protein was decreased by approximately 60%, suggesting cytotoxicity. Cells cultured without insulin were more sensitive to inhibition of 14C-alanine incorporation into proteins by low concentrations of TNF-alpha compared with cells cultured with insulin. These results suggest that TNF-alpha affects glucose metabolism, causing increased basal uptake in muscle cells and decreased uptake in adipocytes. TNF-alpha appears to affect general cell metabolism, including glucose transport in adipocytes, and not specifically insulin-stimulated glucose transport.