Haemophilus influenzae penetrates between epithelial cells via an unknown mechanism. A chromosomal library of nonencapsulated H. influenzae strain A960053 DNA was constructed in Escherichia coli DH5 to identify bacterial genes contributing to this paracytosis. Two E. coli clones that contained open reading frames (ORFs) homologous to HI0636 to HI0641 of H. influenzae strain Rd and that showed an increased penetration in epithelial cell layers of the human bronchial epithelial cell line NCI-H292 were identified. ORFs HI0636 and HI0638, encoding two small proteins of unknown functions, were further investigated. The clone containing ORFs HI0636 and HI0637 as well as the clone containing ORF HI0638 showed a significant increase in penetration. Disruption of HI0638 by kanamycin box insertion in H. influenzae strain A960053 resulted in loss of penetration into the epithelial cell layers. Disruption of HI0636 had no effect on penetration in this model system. Since a role for HI0637 in the paracytosis of H. influenzae is very unlikely because it encodes TrpS, we conclude that the protein encoded by ORF HI0638 may function as a paracytin, while that encoded by HI0636 may have an auxiliary function.