Using a THP-1 human promonocyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype, we previously showed that tolerant cells remain responsive to LPS endotoxin with degradation of IkappaB in the cytosol and nuclear translocation and accumulation of p50 and p65 NF-kappaB transcription factors. Despite this, endotoxin-inducible NF-kappaB-dependent innate immunity genes, like IL-1beta, remained transcriptionally unresponsive in the tolerant phenotype, similar to the endotoxin tolerance observed in sepsis patients. In this study, we examined this paradox and found that RelB, another member of the NF-kappaB family, is induced during the establishment of tolerance. RelB expression correlated with IL-1beta repression, and sepsis patients showed increased RelB when compared with normal controls. Transient expression of RelB inhibited IL-1beta in endotoxin-responsive cells. In the inverse experiment, small inhibitory RNAs decreased RelB expression in tolerant cells and restored endotoxin induction of IL-1beta. When we examined tolerant cell extracts, we found transcriptionally inactive NF-kappaB p65/RelB heterodimers. Taken together, our findings demonstrate that RelB can repress proinflammatory gene expression, and suggest that RelB expression in sepsis patient blood leukocytes may play a role in the endotoxin-tolerant phenotype.