Escherichia coli K1 is an important neonatal pathogen that is usually transferred from maternal to infant gastrointestinal tract at the time of parturition. Approximately 20% of neonates are colonized, and a proportion of colonized infants goes on to have systemic infection. Entry into the bloodstream from the gastrointestinal tract is hypothesized to occur via epithelial cell invasion. Invasion of multiple epithelial cell lines was studied using gentamicin protection assays and transcytosis of polarized monolayers. Electron microscopy was used to confirm cellular invasion. Cell lines used include two human gastrointestinal lines, Caco-2 and T84; a human respiratory cell line, A549; a human laryngeal cell line, HEp-2; and a canine kidney cell line, MDCK. A virulent E. coli K1 strain, RS218, readily invaded HEp-2, A549, and T84 cell lines in gentamicin protection assays, but was less invasive into MDCK and Caco-2 cells. RS218 also demonstrated transcytosis of both T84 and Caco-2 cells. Four clinical isolates of E. coli K1 demonstrated levels of transcytosis of T84 cells similar to RS218. Caco-2 invasiveness correlated with length of time in tissue culture with maximum invasiveness demonstrated at 11 d in culture, when cells were polarized and differentiated.