Escherichia coli is the most common gram-negative bacteria causing meningitis during the neonatal period, but is unclear what microbial factors mediate traversal of E. coli across the blood-brain barrier. Outer membrane protein A (OmpA), a highly conserved 35-kDa protein, was examined for its role in E. coli K1 invasion of brain microvascular endothelial cells (BMEC). The invasive capability of the OmpA+ strains was 25- to 50-fold greater than that of OmpA- strains, and the invasive capability of OmpA- strains was restored to the level of the OmpA+ strain by complementation with the OmpA+ E. coli into BMEC. Two short synthetic peptides (a hexamer, Asn-27-Glu-32, and a pentamer, Gly-65-Asn-69) generated from the N-terminal amino acid sequence of OmpA exhibited significant inhibition of OmpA+ E. coli invasion, suggesting that these two sequences represent the OmpA domains involved in E. coli invasion of BMEC. These findings suggest that OmpA is the first microbial structure identified to enhance E. coli invasion of BMEC, an important event in the pathogenesis of E. coli meningitis.