Melatonin has been proposed as regulating the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. To further investigate the melatonin's role in IL-2/IL-2R system, we established an inducible T-REx expression system in Jurkat cells in which the protein levels of HIOMT enzyme or MT(1) receptor were significantly down-regulated upon tetracycline incubation. We found that T-REx Jurkat cells with lower levels of HIOMT activity, and consequently lower content of endogenous melatonin, showed IL-2 production decrease after activation with lectin. Likewise, tetracycline-inducible stable cell line expressing MT(1) antisense produced decreased amounts of IL-2 (mRNA and protein levels) after stimulation. Moreover, in T-Rex-MT(1) cells incubated with tetracycline, a sub-optimal PHA dose failed to induce the early activation marker CD25 on the cell surface. The results shown here support the relevance of endogenous melatonin and its signaling in T cell activation.