The family of NF-kappaB transcription factors essentially regulates immune-related gene expression. Recently, we isolated and characterized the classical NF-kappaB/inhibitor kappaB (IkappaB) homologues from a "living fossil," the horseshoe crab, Carcinoscorpius rotundicauda. Interestingly, this ancient species also harbors another class I NF-kappaB p100 homologue, C. rotundicauda Relish (CrRelish). Similar to Drosophila Relish and the mammalian p100, CrRelish contains both the Rel-homology domains (RHD) and the IkappaB-like domain. In this study, we found that the RHD of CrRelish can recognize horseshoe crab and human kappaB response elements and activate the downstream reporter in vitro, thereby suggesting the evolutionary conservation of this molecule. Pseudomonas aeruginosa infection transcriptionally upregulates CrRelish, which exhibits a dynamic protein profile over the time course of infection. Surprisingly, secondary infection reinduced an upsurge in CrRelish protein expression to a level which overrode the protein degradation at 12 h postinfection. These observations strongly suggest the involvement of CrRelish in antibacterial defense. Secondary infection causes (i) the maintenance of a favorable expression-competent sequence context of the CrRelish gene and/or (ii) the derepression or stabilization of the CrRelish transcript resulting from the primary infection to enable the more rapid expression and accumulation of the CrRelish protein, reflecting apparent signal/immune priming in a repeated infection.