We performed radioimmunoprecipitation followed by serial immunoblots to show that, in the unstimulated Jurkat T cell line, the NF-kappa B/Rel family proteins, p80-c-Rel, p105-NF-kappa B, p65-NF-kappa B, p50-NF-kappa B and p36-I kappa B alpha, can be detected as complexes using antisera against c-Rel, p105-NF-kappa B or p65-NF-kappa B. p36-I kappa B alpha and p105, both known inhibitors of NF-kappa B function, can physically associate with NF-kappa B/Rel family members, but not with each other. In vivo and in vitro phosphorylation experiments demonstrated that NF-kappa B/Rel family members, including p105, c-Rel, p50, p65 (for the first time for p50 and p65) and p36-I kappa B alpha are also phosphoproteins. Phosphoserine and phosphothreonine residues were identified in these proteins isolated from unstimulated Jurkat cells. Both unphosphorylated and hyperphosphorylated forms of p36-I kappa B alpha were found in the complexes, suggesting that hyperphosphorylated I kappa B alpha is still capable of associating with the NF-kappa B/Rel family members. After stimulation with phorbol 12-myristate 13-acetate and phytohaemagglutinin for 10 min, p105-NF-kappa B and p50-NF-kappa B, but not p36-I kappa B, were highly phosphorylated. Phosphopeptide mapping of p105 showed that phorbol ester/phytohaemagglutinin stimulation may change p105 phosphorylation qualitatively.