Arbovirus infection of cerebral microvascular endothelial cells was investigated in an in vitro mouse brain endothelial (MBE) cell model. Alphaviruses replicated to a greater extent than did flaviviruses, indicating that viremia may be important in neuroinvasion. Also, some viruses (e.g., Semliki Forest virus) replicated to high titers while others (e.g., Murray Valley encephalitis virus) did not. Viruses that replicated to high titers showed luminal polarity of virus release, indicating that infection of the endothelium may be important in both maintenance of viremia and neuroinvasion; viruses that did not so replicate showed abluminal polarity of release, indicating that virus is actively transported across the endothelial monolayer, another mechanism for neuroinvasion. Infection of MBE cells with highly and less-neuroinvasive strains achieved similar results, indicating that the endothelium does not discriminate between neuroinvasive strains. However, the cerebral microvascular endothelium may be important in discriminating between viruses that invade the brain parenchyma and those that do not: neuron-tropic Ross River virus T48 replicated to higher titers than did ependymal-tropic Ross River virus NB5092. Thus, viruses probably use multiple cellular mechanisms to invade the central nervous system across the cerebral microvascular endothelium.