Cytokine release from inflammatory (CD14⁺) cells is reduced after repeated stimulation with lipopolysaccharide (LPS; LPS tolerance). However, it is not known whether LPS tolerance can be induced in CD14⁻ cells. The aim of the present study was to determine whether endothelial cells [human umbilical vein endothelial cells (HUVEC)] could be rendered tolerant to LPS with respect to LPS-induced polymorphonuclear neutrophil (PMN) adhesion. LPS stimulation (0.5 μg/ml; 4 h) of naive HUVEC increased PMN adhesion. Pretreatment of HUVEC with LPS (0.5 μg/ml) for 24 h resulted in a reduction in the proadhesive effects of a subsequent LPS challenge. The initial LPS stimulation increased 1) mobilization of the nuclear transcription factor NF-κB to the nucleus and 2) surface levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and E-selectin. In LPS-tolerant HUVEC, a second LPS challenge resulted in 1) less accumulation of NF-κB in the nucleus, 2) a reduction in E-selectin expression, and 3) unchanged ICAM-1 expression. LPS-tolerant cells were still capable of mobilizing NF-κB in response to stimulation with either interleukin-1β or tumor necrosis factor-α, resulting in elevated E-selectin levels and increased PMN adhesion. These studies show for the first time that LPS tolerance can be induced in endothelial cells with respect to PMN adhesion. This tolerance is specific for LPS and is associated with an inability of LPS to mobilize NF-κB, resulting in less E-selectin expression.