Among the multiple mechanisms that control the intensity and duration of macrophage activation, the development of a state of refractoriness to a second stimulation in cells treated with lipopolysaccharide (LPS) has long been recognized. Release of inhibitory cytokines and alterations in intracellular signaling pathways may be involved in the development of LPS tolerance. Although a number of molecules have been implicated, a detailed picture of the molecular changes in LPS tolerance is still missing. We have used a genome-wide gene expression analysis approach to (i) define which fraction of LPS target genes are subject to tolerance induction and (ii) identify genes that are expressed at high levels in tolerant macrophages. Our data show that in LPS-tolerant macrophages the vast majority of LPS-induced gene expression is abrogated. The extent of tolerance induction varies for individual genes, and a small subset of genes appears to be exempted. Compared to other negative control mechanisms of macrophages, e.g., IL-10-induced deactivation, LPS tolerance inhibits a much wider range of transcriptional targets. Some previously described negative regulators of TLR signaling (e.g. IRAK-M) were confirmed as expressed at higher levels in LPS-tolerant macrophages. In addition, we discuss other potential players in LPS tolerance identified in this group of genes.