Monocyte exposure to LPS induces a transient state in which these cells are refractory to further endotoxin stimulation. This phenomenon, termed endotoxin tolerance (ET), is characterized by a decreased production of cytokines in response to the proinflammatory stimulus. We have established a robust model of ET and have determined the time frame and features of LPS unresponsiveness in cultured human monocytes. A large number of genes transcribed in tolerant monocytes were classified as either “tolerizable” or “nontolerizable” depending on their expression levels during the ET phase. Tolerant monocytes exhibit rapid IL-1R-associated kinase-M (IRAK-M) overexpression, high levels of triggering receptor expressed on myeloid cells-1 (TREM-1) and CD64, and a marked down-regulation of MHC molecules and NF-κB2. These cells combine potent phagocytic activity with impaired capability for Ag presentation. We also show that circulating monocytes isolated from cystic fibrosis patients share all the determinants that characterize cells locked in an ET state. These findings identify a new mechanism that contributes to impaired inflammation in cystic fibrosis patients despite a high frequency of infections. Our results indicate that a tolerant phenotype interferes with timing, efficiency, and outcome of the innate immune responses against bacterial infections.