Synthetic analogs of vitamin D induce apoptosis in cultured breast cancer cells and cause regression of experimentally-induced rat mammary tumors. To further elucidate the mechanisms involved, we have examined interactions between two vitamin D analogs (CB1093 and EB1089) and known mediators of apoptosis, TNFalpha and ceramide. Pretreatment of MCF-7 breast cancer cells with CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFalpha as assessed by cell viability assay, DNA fragmentation and videomicroscopy. No significant changes in the levels of TNFalpha or TNF-RI transcripts were detected. CB1093 primed cells demonstrated enhanced responsiveness to cell permeable C2-ceramide in terms of increased DNA fragmentation and loss of cell viability. Activation of cytosolic phospholipase A2 (cPLA2) has been implicated in TNFalpha-mediated apoptosis. As assessed by [3H]-arachidonic acid release, cells primed for 48 h with CB1093 (50 nM) showed enhanced cPLA2 activation in response to TNFalpha or ceramide. CB1093 treatment alone led to cPLA2 activation and loss of cell viability which was inhibited by the specific inhibitor AACOCF3. These results suggest that TNFalpha and vitamin D analogs share a common pathway leading to apoptosis involving cPLA2 activation and/or ceramide generation.