Proteins possessing the caspase recruitment domain (CARD) motif have been implicated in pathways leading to activation of caspases or NF-kappaB in the context of apoptosis or inflammation, respectively. Here we report the identification of a novel protein, CARDINAL, that contains a CARD motif and also exhibits a high degree of homology to the C terminus of DEFCAP/NAC, a recently described member of the Apaf-1/Nod-1 family. In contrast with the majority of CARD proteins described to date, CARDINAL failed to promote apoptosis or NF-kappaB activation. Rather, CARDINAL potently suppressed NF-kappaB activation associated with overexpression of TRAIL-R1, TRAIL-R2, RIP, RICK, Bcl10, and TRADD, or through ligand-induced stimulation of the interleukin-1 or tumor necrosis factor receptors. Co-immunoprecipitation experiments revealed that CARDINAL interacts with the regulatory subunit of the IkappaB kinase (IKK) complex, IKKgamma (NEMO), providing a molecular basis for CARDINAL function. Thus, CARDINAL is a novel regulator of NF-kappaB activation in the context of pro-inflammatory signals.