Previous studies from this laboratory as well as others have demonstrated that breast tumor cells fail to undergo primary apoptosis in response to agents which induce DNA damage such as ionizing radiation and the topoisomerase II inhibitor adriamycin. Similarly, the primary response of breast tumor cells to vitamin D(3) [1,25-(OH)(2)-D(3)] and its analogs such as EB 1089 is growth inhibition, with apoptosis occurring in only a small fraction of the cell population. The possibility that the combination of vitamin D(3) compounds with radiation might promote cell death (i.e. through a differentiation stimulus plus DNA damage) was investigated by exposing both TP53 (formerly known as p53) wild-type and TP53 mutated breast tumor cells to 1,25-(OH)(2)-D(3) or EB 1089 for 48 h prior to irradiation. This combination resulted in enhanced antiproliferative effects in the TP53 wild-type MCF-7 cells based on both a clonogenic assay and the determination of numbers of viable cells. The combination of EB 1089 with radiation increased DNA fragmentation based on both the terminal transferase end-labeling (TUNEL) and bisbenzamide spectrofluorometric assays, suggesting the promotion of apoptosis. The observed increase in DNA fragmentation was not due to an enhancement of the extent of initial DNA damage induced by radiation. These findings suggest that vitamin D compounds may be useful in combination with radiation in the treatment of breast cancer.