Apoptosis induced by ligation of either the tumor necrosis factor (TNF) p55 receptor or the Fas/APO-1/CD95 receptor has been suggested to require ceramide as a signaling molecule. Ceramide is formed as a result of sphingomyelinase (SMase) activation in the sphingomyelin cycle, and ligation of TNF and Fas receptors has been reported to stimulate SMase activity. We have studied the effects of D609, a xanthogenate compound with antitumoral properties, on TNF- or Fas-induced apoptosis of monocytic U937 cells. First, the effects of D609 on SMase activity were assessed using in vitro assays for neutral and acidic SMase, and the results suggested that D609 caused a modest stimulation of the activity of both SMases in U937 cells. Exposure of U937 cells for 6 h to TNF or anti-Fas mAb induced apoptosis in 40-45% of the cells, as measured by fluorescent staining of nuclear chromatin. Cotreatment with D609 potentiated TNF- as well as Fas-mediated apoptosis up to 70 and 90%, respectively. Furthermore, in incubations with D609 alone, 60% of the cells became apoptotic within 16 h. Since D609 has been reported to inhibit protein kinase C (PKC) activity, the effect of phorbol 12-myristate 13-acetate (PMA) on D609-induced apoptosis was investigated. PMA was found to inhibit D609-induced apoptosis in U937 cells as well as cell death induced by TNF and anti-Fas mAb. Thus, PKC inactivation may play an important role in the regulation of apoptosis in U937 cells. In summary, the present results show that D609 stimulates SMase activity, potentiates TNF- and Fas-induced apoptosis, and induces apoptosis on its own in U937 cells.