Knowledge about the in vivo role of endothelium in chronic human atherosclerosis has mostly been derived by insights from mouse models. Therefore, we set out to establish by microarray analyses the gene expression profiles of endothelium from human large arteries, as isolated by laser microbeam microdissection, having focal atherosclerosis of the early or the advanced stage. Within individual arteries, the endothelial transcriptomes of the lesional and unaffected sides were compared pairwise, thus limiting genetic and environmental confounders. Specific endothelial signature gene sets were identified with changed expression levels in either early (n = 718) or advanced atherosclerosis (n = 403), relative to their paired plaque-free controls. Gene set enrichment analysis identified distinct sets of chemokines and differential enrichments of nuclear factor-kappaB-, p53-, and transforming growth factor-beta-related genes in advanced plaques. Immunohistochemistry validated the discriminative value of corresponding endothelial protein expression between early (fractalkine/CX3CL1, IP10/CCL10, TBX18) or advanced (BAX, NFKB2) stages of atherosclerosis and versus their plaque-free controls. The functional involvement of transforming growth factor-beta signaling in directing its downstream gene repertoire was substantiated by a consistent detection of activated SMAD2 in advanced lesions. Thus, we identified truly common, local molecular denominators of pathological changes to vascular endothelium, with a marked distinction of endothelial phenotype between early and advanced plaques.