A long term treatment of T cells with tumor necrosis factor alpha (TNF-α) paradoxically inhibits the immunologic responses to TCR/CD3 stimulation. The voltage-gated K+ channels (Kv) of T cells attracted attention as a pharmacological target for the treatment of autoimmune diseases. Here, the authors investigated the effects of TNF-α on the Kv current (IKv) and its upregulation by CD3 in human T cells. Acute treatment with TNF-α (10 min) temporarily decreased IKv in Jurkat-T cells (cells subsequently recovered after treatment >12 h), whereas CD3 stimulation for 24 h increased IKv amplitude more than two-fold. Furthermore, chronic pretreatment with TNF-α almost completely blocked the IKv increase induced by CD3 stimulation. An immunoblot study confirmed an increase in the protein level of Kv induced by CD3 stimulation, and its inhibition by TNF-α pretreatment. In addition, the facilitation of IKv by CD3 stimulation and its inhibition by pretreatment with TNF-α were confirmed in freshly isolated human peripheral CD4(+) T cells, in which the voltage-dependence of IKv was unaffected by TNF-α and/or CD3 stimulation. We conclude that the inhibition of CD3-induced Kv upregulation by TNF-α might be associated with the paradoxical suppression of T cell function by TNF-α under conditions of chronic inflammation.