Nuclear factor (NF)-kappa B proteins regulate the transcription of numerous genes involved in the immune response, transcription control, and viral pathogenesis. To examine the effect of ectopic expression of NF-kappa B proteins on DNA-binding activity and gene expression, individual NF-kappa B subunit genes were introduced into NIH 3T3 cells via retrovirus-mediated gene transfer. Expression of NF-kappa B subunits RelA (p65), NF-kappa B1 (p105), NF-kappa B2 (p100), and c-Rel increased the basal level of nuclear NF-kappa B DNA binding in NIH 3T3 cells, whereas expression of delta RelA (p65 delta) and NF-kappa B2 (p52) subunits did not affect basal level activity. Tumor necrosis factor-alpha treatment of the NF-kappa B-expressing cells stimulated the induced level of DNA-binding activity, reflecting interaction between endogenous murine and transfected human NF-kappa B proteins. Interestingly, expression of RelA (p65), c-Rel, NF-kappa B1 (p105), NF-kappa B2 (p100), and NF-kappa B2 (p52) subunits increased I kappa B alpha protein levels from 3- to 30-fold, indicating that one mechanism to compensate for the increased expression of NF-kappa B proto-oncogenes was to increase the synthesis and/or stability of the regulatory I kappa B alpha protein. In addition, overexpression of RelA (p65), c-Rel, NF-kappa B2 (p100), and NF-kappa B2 (p52) altered the induction kinetics of IFN-beta mRNA after Sendai virus infection, whereas overexpression of NF-kappa B1 (p105) dramatically decreased IFN-beta mRNA induction.