Interferons (IFNs) and retinoic acid (RA) are known to possess antiproliferative effects in various human cancer cell lines, including squamous cervix carcinoma and breast cancer cell lines. Frequently synergistic effects could be observed by the combination of both, and in clinical trials high response rates could be achieved by IFN-alpha in combination with 13-cis-RA in patients with squamous carcinoma of the uterine cervix. Since radiation-potentiating effects of IFNs and RA were described, we evaluated the additional impact of irradiation on three human breast cancer cell lines and investigated the antiproliferative effects of single, double, or triple treatments with IFN-gamma, 9-cis-RA, and irradiation. Antiproliferative effects were observed in all cell lines by any single treatment. When combining IFN-gamma with 9-cis-RA, synergism could be observed in all experiments. The combination of either IFN-gamma or 9-cis-RA with irradiation resulted mostly in additive effects. Irradiation contributed additive or further synergistic effects to the already synergistic effects of the combination of these two drugs. Thus synergism of IFN-gamma and 9-cis-RA always at least persisted fully. These results suggest that a regimen of IFN, RA, and radiotherapy (RT) might be a promising combination in the therapy of solid tumors, where RT is part of the conventional treatment.