Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features among 44 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates while the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast with the productive infection of cytolytic EVs (e.g. echoviruses 6 and 30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes, and reorganization of the mitochondrion network in a small cluster near the perinuclear space.