The incubation of human mammary adenocarcinoma cells (BT-20) with tumor necrosis factor alpha in the absence or presence of cycloheximide resulted in progressive DNA fragmentation. This was preceded by a sustained increase in intracellular free Ca2+ concentration and was not detected in cells pretreated with intracellular Ca2+ chelators, calmodulin antagonists, or activators of protein kinase C. Image analysis of fura-2-loaded BT-20 cells treated with tumor necrosis factor alpha revealed that, in many cells, the initial increase in Ca2+ level occurred in a cellular region that corresponded to the localization of the nucleus. Our findings suggest that tumor necrosis factor alpha can promote an increase in intranuclear free Ca2+ which, in turn, may stimulate Ca(2+)-dependent endonuclease activity, resulting in DNA fragmentation and apoptosis.