All-trans-retinoic acid and the tumor suppressor promyelocytic leukemia protein (PML) are potent regulators of the growth of cancer cells. This study investigates the individual and combined effects of PML, when overexpressed by the recombinant PML adenovirus, and all-trans-retinoic acid on the proliferation of human estrogen-receptor negative SKBR-3 and estrogen-receptor positive MCF-7 breast cancer cell lines. All-trans-retinoic acid caused a significant degree of cell death in SKBR-3 cells and MCF-7 cells, and PML elicited a similar incidence of or slightly more cell death in MCF-7 cells. Dual-treated cells displayed significantly less cell death than did single-treated cells in the same cell line. We concluded that PML and all-trans-retinoic acid cause cell death by different pathways: PML activates ERK1/2, p38 MAPK, and p21; arrests the cell cycle; and later causes cell death; and all-trans-retinoic acid activates proteasome function, caspase cleavage, and apoptosis. The combined use of all-trans-retinoic acid and PML gene therapy may not be the best treatment for patients with cancer, because the ubiquitinylation of PML and its subsequent proteasome-dependent degradation by retinoic acids occur before overexpressed PML exhibits tumor-suppressive activity.