Ubiquitination-mediated degradation of the RelA subunit of nuclear factor-kappaB (NF-kappaB) is critical for the termination of NF-kappaB activation. However, the precise mechanism for the ubiquitination of RelA is still not fully understood. Here we report that tumor necrosis factor-alpha (TNFalpha) induces RelA polyubiquitination at the lysine 195 residue, and this ubiquitination event is critical for the degradation of RelA and termination of TNFalpha-mediated NF-kappaB activation. Overexpression of a RelA mutant with an arginine substitution for the lysine 195 residue dramatically inhibits RelA polyubiquitination and induces a stronger NF-kappaB activation compared with the wild type. Reconstitution of RelA-deficient mouse embryo fibroblast cells with wild-type RelA or RelA containing a K195R mutation revealed the importance of this site in TNFalpha-mediated RelA polyubiquitination, degradation, and attenuation of NF-kappaB activation. Our finding is the first report that substitution of a key RelA lysine residue with arginine inhibits TNFalpha-induced RelA ubiquitination and enhances TNFalpha-induced NF-kappaB activation.