Cross-modulation between androgen receptor (AR) and NF-kappaB/Rel proteins was studied using various androgen- and NF-kappaB-regulated reporter genes under transient transfection conditions. In COS-1 cells, elevated expression of RelA (p65) repressed AR-mediated transactivation in a dose-dependent manner, whereas NFkappaB1 (p50), another major member of the NF-kappaB family, did not influence transactivation. The repression of AR appeared to involve the N-terminal region of the protein between residue 297 and the DNA-binding domain. RelA-mediated transrepression could not be overcome by increasing the amount of AR. Transcriptional interference between RelA and AR was mutual in that cotransfected AR was able to attenuate transactivation by RelA in a dose- and steroid-dependent fashion. An excess of RelA was able to rescue the repression to some extent. Immunological analyses of RelA and AR protein levels indicated that transrepression was not due to reciprocal decrease in their amounts. Neither did AR increase the concentration of IkappaBalpha, which can sequester and inactivate RelA. Electrophoretic mobility shift assays using extracts from cotransfected cells and purified recombinant proteins showed that AR and RelA did not significantly influence each other's DNA binding activity. Nevertheless, protein-protein interaction experiments demonstrated a weak association between AR and RelA. Collectively, these data suggest that the mutual repression in intact cells is due to formation of AR-RelA complexes that are held together by another partner or to competition for a coactivator required for transcription.