The abilities of Escherichia coli to induce diarrhea by enterotoxin production and by intestinal cell penetration have not been explored simultaneously in children with diarrheal disease. In this study, we investigated 36 infants and children with acute diarrhea and 17 healthy controls. From each patient's rectal swab culture, E coli colonies were tested for enterotoxin production by intragastric inoculation in suckling mice and screened for cell penetration with HEp-2 cells. Colonies showing invasiveness in HEp-2 cells were further tested by inoculation into guinea pig conjunctivae. Salmonella or Shigella strains were isolated from 42% of the patients with diarrhea. Enterotoxin-producing strains of E coli were found in 86% of the diarrhea group and in 41% of controls. Strains with capability to invade epithelial cells were found in 30% of the diarrhea group and in 12% of controls. In seven patients, E coli strains demonstrated both enterotoxin production and invasiveness. In an overall view, considering Salmonella, Shigella, and enteroinvasive or enterotoxigenic E coli, a possible etiologic agent was demonstrated in 94% of infants with diarrheal disease. However, the high frequency of enterotoxigenic strains in healthy controls suggest that in some patients with diarrhea there may be a coincidental carrier state with diarrhea actually due to another cause.