We have shown that melatonin exerts a prooxidant activity in U937 cells, a tumor human promonocytic cell line. (1) Here we show that melatonin induces a strong canonical activation of NF-kappaB, inducing IkappaBalpha degradation and the consequential nuclear translocation of p50/p65 subunits. The timing of NF-kappaB activation overlaps with the timing of reactive oxygen species (ROS) production due to melatonin. Overexpression of dominant-negative IkappaB, which prevents a possible NF-kappaB activation, transformed melatonin in a proapoptotic molecule. These data indicate for the first time that melatonin can trigger NF-kappaB activation and might suggest a possible role for ROS induced by melatonin. Results indicate a possible involvement in the survival pathway of melatonin-generated ROS as secondary messengers.