Interferons inhibit cell growth in normal and tumor-derived cells. The molecular basis of interferons antiproliferative activity remains to be defined. Using subtraction hybridization, a human melanoma differentiation associated gene, mda-20, has been identified that is down-regulated by treatment with interferon. Sequence analysis indicates that mda-20 is human ribosomal protein L23a (rp L23a). The mRNA levels of rp L23a and growth are diminished in a variety of human tumor cell lines following treatment with human fibroblast interferon, interferon-beta (IFN-beta). Expression of rp L23a is also reduced in human melanoma cells treated with human leukocyte (IFN-alpha) and immune (IFN-gamma) interferons, but not by growth inhibition resulting from serum starvation. These findings suggest that growth suppression alone is not sufficient to reduce rp L23a expression. Instead, reduced rp L23a mRNA results from biochemical changes mediated by interferons. Ectopic expression of an antisense rp L23a sequence in human HeLa cervical carcinoma cells results in a reduction in colony formation indicating a direct antiproliferative effect by inhibiting rp L23a expression. The mechanism underlying inhibition in rp L23a expression in IFN-beta-treated cells may involve antisense rp L23a RNA. These results suggest that rp L23a may be one of the target molecules involved in mediating growth inhibition by interferon.