To assess the feasibility of using cDNA microarrays to understand the response of endothelial cells to lipopolysaccharide (LPS) and to evaluate potentially beneficial agents in treatment of septic shock, human umbilical vein endothelial cells were exposed to Escherichia coli LPS for 1, 4, 7, 12, or 24 h. Total RNA was isolated and reverse-transcribed into³³P-labeled cDNA probes that were hybridized to human GeneFilter microarrays containing ∼4,000 genes. The mRNA levels of several genes known to respond to LPS changed after stimulation. In addition, a number of genes not previously implicated in the response of endothelial cells to LPS also appeared to be altered in expression. Nuclear factor-κB (NF-κB) was shown to play an important role in regulating genes identified from the microarray studies. Pretreatment of endothelial cells with a specific NF-κB translocation inhibitor eliminated most of the alterations in gene expression. Quantitative RT-PCR results independently confirmed the microarray results for monocyte chemotactic protein-1 and interleukin-8, and enzyme-linked immunosorbent assays demonstrated that augmented transcription was followed by translation and secretion.