The inhibitor kappa B (I kappa B) protein is an important component of the nuclear factor kB (NF-kappa B) signaling pathway. We have cloned and characterized the I kappa B-alpha homologue gene (named EaI kappa B-alpha) from grouper (Epinephelus akaara), expressed it in Escherichia coli and generated a rabbit polyclonal antibody. EaI kappa B-alpha encodes a 308-amino acid protein with evolutionarily and functionally homology to mammalian I kappa B-alpha. Over-expression of wild-type EaI kappa B-alpha or a mutated, nondegradable EaI kappa B-alpha (EaI kappa B-alpha M) in Escherichia coli both inhibited the degradation of cellular EaI kappa B-alpha, activation of NF-kappa B and expression of tumor necrosis factor alpha (TNF-alpha). EaI kappa B-alpha protein is restricted to the cytoplasm in unstimulated cell. Activated by diverse stimuli, EaI kappa B-alpha is degraded, thereby releasing NF-kappa B into nucleus and activating NF-kappa B-mediated transcripts. EaI kappa B-alpha and other studied NF-kappa B target genes are induced by LPS in kidney and liver, which suggests that EaI kappa B-alpha may play an important role in immune response of grouper.