NF-kappa B is a heterodimeric transcription factor consisting of subunits of 50 kDa (p50) and 65 kDa (p65). cDNA clones encoding the chicken NF-kappa B p65 subunit were isolated. Sequence analysis showed that chicken p65 is approximately 55% identical to the mouse and human p65 proteins, and contains the Rel homology domain (RHD) in its N-terminal 286 amino acids (aa) and the putative transactivation domain in its C-terminal region. The RHD is particularly highly conserved between the chicken and mammalian p65 proteins. Northern blot hybridization analysis detected the expression of a 2.6-kb transcript of p65 in various organs, with the highest level in spleen. A fusion protein containing the RHD of chicken p65 was found to bind to a consensus kappa B-site in an electrophoretic mobility shift assay (EMSA). This binding was specifically inhibited by the presence of fusion proteins containing the C-terminal ankyrin repeats domain (ARD) of chicken p105, the precursor protein for the p50 subunit. Immunoprecipitation analysis showed that p65 formed a complex(es) with multiple cellular proteins, including p50, p105 and c-Rel in chicken spleen cells.