Intestinal bacterial translocation is facilitated in a variety of clinical conditions involving increased intestinal permeability, such as shock and trauma. Because there is both in vivo and in vitro evidence that enteric bacteria can be internalized by intestinal epithelial cells, experiments were designed to test the effect of increased intestinal permeability on enterocyte endocytosis of enteric bacteria. Mature, confluent cultures of HT-29 enterocytes were placed in a calcium-free solution for 1 h. Enterocyte viability was not noticeably altered, but transepithelial electrical resistance was significantly decreased (indicating a decrease in epithelial junctional integrity), and the enterocytes were pulled apart. Electron microscopic observations revealed enteric bacteria preferentially adherent on the exposed enterocyte lateral surface, and the numbers of viable enteric bacteria (Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, Escherichia coli, and Enterococcus faecalis) internalized by these enterocytes were significantly increased. Restoration of calcium restored confluency to enterocyte cultures, and bacterial intemalization reverted to control levels. Thus, calcium-dependent junctional integrity might play a role in augmenting bacterial translocation in clinical conditions associated with increased intestinal permeability.