Routes of alphaviral entry have been studied with the transformed human endothelial vein cell line ECV 304. This cell line can be cultured on tissue culture inserts which permits apical (lumenal) and basolateral (ablumenal) cell surfaces to be infected and observed separately. Semliki Forest Virus (SFV), a prototype alphavirus, was able to infect and replicate from both the apical and basolateral sides. Transcytosis is not the route by which SFV passes the endothelial cell barrier as demonstrated by polarised infection of junctionally tight ECV monolayers in which translation was inhibited. A "grow-through" replication may play a role in SFV pathogenesis. Infected ECV cells produced interfering substances that inhibited viral infection. Higher multiplicities of infection resulted in infection and complete destruction of the monolayer.