Antibody responses generated by mice to the dengue-2 virus NS1 protein (D-2V NS1) were influenced by MHC class II (I-A) haplotype but each antiserum cross-reacted with human fibrinogen, thrombocytes and endothelial cells. To investigate these findings, a highly avid subclone (MAb 1G5.4-Aff1-C3) was selected from a parent hybridoma that secreted a monoclonal antibody (MAb) specific for the native dimeric form of D-2V NS1. When MAb reactions were compared using a panel of overlapping synthetic peptides covering the entire protein sequence, dimer specificity was found to be a weak reaction with multiple ELK-type motifs present in either the positive (E/D-hydrophobic-K/R) or negative (K/R-hydrophobic-D/E) orientations. MAb 1G5.4-Aff1-C3 and highly avid anti-NS1 polyclonal antisera reacted with the NS1 proteins of the four dengue virus serotypes, but only weakly reacted with the NS1 proteins of the other flaviviruses. MAb 1G5.4-Aff1-C3 and several other anti-NS1 MAbs produced haemorrhage in mice, cross-reacted with human fibrinogen, thrombocytes and endothelial cells, with known epitopes or active sites on human clotting factors and integrin/adhesin proteins present on these cells. D-2V NS1 bound to human endothelial cells via a site within its N-terminal region, which led to significantly increased binding of avid anti-NS1 antibodies. These results identified a potential role of both ‘antigenic’ and ‘biochemical’ mimicry in dengue haemorrhagic fever pathogenesis, consistent with clinical data.