The glycolytic enzyme α-enolase is a plasminogen-binding protein that is generally found in the cytosolic compartment. However, α-enolase can also be expressed on cell surfaces following an inflammatory stimulus via an unknown mechanism. We investigated the effects of Streptococcus sanguinis (S. sanguinis) and the sera of patients with Behçet's disease (BD) on the expression and distribution of α-enolase in human dermal microvascular endothelial cells (HDMECs). HDMECs were stimulated with cultured S. sanguinis and the sera of active BD patients. HDMECs incubated for 6, 12 or 24 h were harvested, and the membrane and cytoplasmic fractions of proteins were extracted. The expression and distribution of α-enolase were analyzed using subcellular fractionation and immunoblotting. Subcellular localization of α-enolase was also assessed by immunocytochemistry. S. sanguinis stimulated the expression of α-enolase in the membranous compartment of HDMECs in a dose-dependent manner. This pattern was also observed in HDMECs incubated with BD patients' sera. Although incubation of HDMECs with sera from healthy controls increased membrane expression of α-enolase, incubation with BD sera resulted in earlier and higher expression of this glycoprotein in the cellular membrane of HDMECs. Immunocytochemistry revealed strong immunostaining of α-enolase in the cytoplasm and cytoplasmic membrane of HDMECs incubated with S. sanguinis or BD patients' sera. In conclusions, these results indicate that S. sanguinis infection and the sera of BD patients with active disease are inflammatory stimuli that can induce membranous α-enolase expression in endothelial cells. Membrane-expressed α-enolase could potentially react with anti-α-enolase antibodies in BD patients' sera, resulting in increased inflammation.