Hyphal invasion of blood vessels is a prominent feature of invasive aspergillosis. During invasive pulmonary aspergillosis, Aspergillus fumigatus hyphae invade the abluminal endothelial cell surface, whereas they invade the luminal endothelial cell surface during haematogenous dissemination. We investigated the endothelial cell response to abluminal and luminal infection with A. fumigatus hyphae in vitro. We found that these hyphae invaded the abluminal endothelial cell surface without inducing the formation of endothelial cell pseudopods. Also, the internalized hyphae were surrounded by a loose network of microfilaments. In contrast, A. fumigatus hyphae invaded the luminal endothelial cell surface by inducing by the formation of endothelial cell pseudopods. These endocytosed hyphae were surrounded by a tight network of microfilaments. Abluminal infection induced greater E-selectin, IL-8, tissue factor and TNF-alpha gene expression, but less endothelial cell damage than did luminal infection. Endothelial cell stimulation by infection of either surface was mediated by endothelial cell-derived TNF-alpha, and was not influenced by gliotoxin secreted by A. fumigatus. These differences in the endothelial cell response to abluminal versus luminal infection may contribute to differences in the pathogenesis of invasive versus haematogenously disseminated aspergillosis.