Proteins of the NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] family may serve as critical pathogen-sensing and signal-transducing molecules within the innate immune system. In the present paper, we show that CLAN [CARD (caspase-recruitment domain), LRR (leucine-rich repeat) and NACHT domain-containing protein], a NACHT-containing protein originally demonstrated to bind and activate pro-caspase 1, is also capable of influencing the functions of other members of the NACHT family. Through heterotypic NACHT-domain interactions, CLAN was found to associate with Nod1, Nod2 and NAC [nucleotide-binding domain and CARD-containing protein; NALP1 (NACHT, LRR and PYRIN protein 1)] when co-expressed in HEK-293T (human embryonic kidney) cells. NF-kappaB (nuclear factor kappaB) reporter assays demonstrated that co-expression of either full-length CLAN or the NACHT domain of CLAN significantly inhibited NF-kappaB activation induced by Nod1 or Nod2 overexpression. In addition, co-expression of CLAN or the NACHT domain of CLAN with Nod1 or Nod2 inhibited the ability of these proteins to generate active IL-1beta (interleukin 1beta) through their association with pro-caspase 1. The NACHT domain of CLAN was demonstrated by co-immunoprecipitation experiments to bind all NACHT domains that were tested, including the NACHT domains from CLAN itself, Nod1, Nod2, cryopyrin, NAC, PAN2 [PAAD [pyrin, AIM (absent-in-melanoma), ASC (apoptosis-associated speck-like protein containing a CARD) and death-domain-like]- and NACHT-containing protein] and NAIP (neuronal apoptosis inhibitory protein). Finally, monocyte-expressed CLAN was found to associate with Nod2 following exposure to bacterial peptidoglycan, implying a regulatory role for interaction of these NACHT proteins in the innate immune response. These studies suggest that by mediating hetero-oligomerization, NACHT domains provide a means by which various NACHT-containing proteins may interact, creating protein-interaction networks that potentially modulate immune responses to invading pathogens.