It has been reported that the cytotoxic effect of tumor necrosis factor (TNF) on cells of several tumor cell lines was potentiated in culture media lacking glucose. Also, the antitumor effect of TNF was shown to be enhanced in vivo in mice treated with insulin to reduce their blood glucose level. The present study was aimed to reveal whether (a) the administration of the glucose antimetabolite 2-deoxy-D-glucose (2DG) has an effect similar to that of reduction of the extracellular glucose concentration; (b) the combined treatment with TNF and 2DG, similar to TNF alone, leads to apoptosis; and (c) there is a preference of cells in a particular phase of the cell cycle to undergo apoptosis in the presence of these agents. Exponentially growing human histiocytic lymphoma U937 cells were exposed to 0.1-0.5 nM of recombinant human TNF-alpha in the absence and presence of 1.0-5.0 mM 2DG. Analysis of the cell proliferation rates and their viability revealed that cytotoxicity of TNF was markedly potentiated by 2DG. Thus, administration of 1.0 mM 2DG to the cultures treated with 0.3 nM recombinant human TNF-alpha increased by 2-3-fold the percentage of dead cells after 24-72 h. The antimetabolite alone, at that low concentration, showed minimal cytotoxicity. More than additive cytotoxic effects also were seen at 2.5 and 5.0 mM concentrations of 2DG. Apoptosis was identified by typical changes in cell morphology, preferential degradation of internucleosomal DNA, and in situ extensive DNA strand breakage. The number of cells with DNA strand breaks after 24-h incubation was increased from 13% (0.1 nM TNF alone) to 20 or 45% in the presence of 2.5 or 5.0 mM 2DG, respectively. There was no evidence of a significant cell cycle phase preference in induction of apoptosis by combined treatment with recombinant human TNF-alpha and 2DG, although 2DG alone reduced the percentage of cells in S and G2 + M, apparently by arresting cells in G1. These data, along with observations in other cell systems, suggest that simultaneous stimulatory signals for growth induction, presumed to be provided by TNF, and growth suppression (inhibition of glycolysis) may preferentially trigger apoptosis of transformed cells. The data also suggest that 2DG may be an effective adjunct to TNF in the clinic, increasing the antitumor potency of this cytokine.