I kappa B-alpha inhibits both DNA-binding and nuclear translocation of dimeric Rel complexes that contain either the RelA or c-Rel proteins. These inhibitory functions of I kappa B-alpha proteins are regulated by both constitutive and inducible phosphorylation. We have mapped the constitutive phosphorylation sites of p40, the avian I kappa B-alpha protein, to a C-terminal acidic serine-rich region that contains four serine residues. Deletions or point mutations that significantly alter the overall negatively charged character of this region abolish association of p40 with Rel proteins in vitro. Serine-to-alanine amino acid substitutions in this region modulate the association of p40 with Rel proteins in vitro and abolish p40-mediated inhibition of DNA-binding by c-Rel. Substitution of aspartic acid residues for the phosphorylated serine residues has no effect on p40-mediated inhibition of DNA-binding. In contrast, the C-terminal acidic serine-rich region is not required for p40-mediated inhibition of nuclear translocation of Rel proteins. Our results demonstrate that p40-mediated inhibition of nuclear translocation and inhibition of DNA-binding by Rel proteins are separable functions. Our results suggest that the phosphorylation status of C-terminal serine residues of I kappa B-alpha proteins will be an important aspect of the autoregulatory feedback loop that enforces temporal control of Rel-regulated gene expression.