The molecular signaling events involved in the inhibition of breast cancer cell growth by retinoic acid and interferon-alpha were investigated. All-trans-retinoic acid and interferon-alpha acted synergistically to inhibit growth of both the estrogen receptor-positive breast cancer cell line MCF-7 and the estrogen receptor-negative line BT-20. In MCF-7 cells, all-trans-retinoic acid potentiated the effects of interferon-alpha by up-regulating the expression of the RNA-dependent protein kinase (PKR). Consequently, the synergism between all-trans-retinoic acid and interferon-alpha down-regulated the expression of c-Myc, but not its functional partner, Max. Transfection of MCF-7 cells with a dominant-negative mutant of PKR relieved c-Myc down-regulation and cell growth inhibition, indicating that PKR is directly involved in c-Myc down-regulation and that c-Myc down-regulation is responsible for the inhibition of cell growth. Corresponding with c-Myc down-regulation, c-Myc.Max heterodimers bound to their consensus DNA sequence were undetectable in cells treated with all-trans-retinoic acid and interferon-alpha, indicating diminished c-Myc functionality. When c-Myc was overexpressed ectopically via a c-Myc expression vector, MCF-7 cells became resistant to growth inhibition by all-trans-retinoic acid plus interferon-alpha. These experiments define the following pathway as a major pathway in the synergistic growth inhibition of MCF-7 cells by all-trans-retinoic acid plus interferon-alpha: all-trans-retinoic acid + interferon-alpha --> upward arrow double-stranded RNA-dependent protein kinase --> downward arrow c-Myc --> cell growth inhibition.