T₄, which is a major secretory product of the thyroid gland, needs to be converted to T₃ by iodothyronine deiodinase to exert its biological activity. After the molecular cloning of human type II iodothyronine deiodinase (DII) complementary DNA, DII expression was unexpectedly detected in human skeletal muscle tissue. In the present study, we have identified DII activity and DII messenger ribonucleic acid (mRNA) in cultured human skeletal muscle cells and studied the mechanisms involved in the regulation of DII expression in those cells. All of the characteristics of the deiodinating activity in cultured human skeletal muscle cells were compatible with those of DII. Northern analysis has demonstrated that DII mRNA, approximately 7.5 kb in size, was expressed in cultured human skeletal muscle cells. DII mRNA and DII activity were rapidly increased by (Bu)₂cAMP, forskolin, or β-adrenergic agonists and were negatively regulated by thyroid hormones in cultured human skeletal muscle cells. Although interleukin-1β and interleukin-6 did not decrease DII expression in cultured human skeletal muscle cells, tumor necrosis factor-α decreased DII expression in those cells in a dose-dependent manner. These data have demonstrated, for the first time, that DII activity and DII mRNA are present in cultured human skeletal muscle cells, and that the DII expression is stimulated byβ -adrenergic mechanisms through a cAMP-mediated pathway and is negatively regulated by thyroid hormones and tumor necrosis factor-α.