Pellino is a Drosophila protein originally isolated in a two-hybrid screen for proteins interacting with the serine/threonine kinase, pelle. Although mammalian homologs have been identified in mouse and man, the function of pellino is as yet unknown. In this study, the cloning, expression pattern, and a preliminary characterization of mouse pellino-2 is described. These studies reveal that mouse pellino-2 is expressed during embryogenesis and in a tissue-restricted manner in the adult. IL-1 induces the association of mouse pellino-2 with the mouse pelle-like kinase/IL-1R-associated kinase protein, a mammalian homolog of pelle. Ectopic pellino-2 expression did not result in NF-κB activation. However, ectopic expression of a mouse pellino-2 antisense construct inhibited IL-1 or LPS-induced activation of NF-κB-dependent IL-8 promoter activity. Our data reveal that mouse pellino-2 is a tissue-restricted component of a signaling pathway that couples the mouse pelle-like kinase/IL-1R-associated kinase protein to IL-1-or LPS-dependent signaling.