Histopathological evidence suggests that the adherence of enteropathogenic Escherichia coli (EPEC) to the mucosa of the small bowel is an important step in pathogenesis. Several reports have shown that many EPEC isolates adhere to HEp-2 and HeLa cells in tissue cultures. In the HeLa cell assay, there are at least two distinct patterns of adherence: localized adherence, which is characterized by the formation of bacterial microcolonies, and diffuse adherence, in which bacteria cover the cell uniformly. We have found that these two patterns can be demonstrated in HEp-2 cells as well as in HeLa cells and that the results of the two assays are closely correlated. Using a DNA probe which is sensitive and specific for localized adherence to HEp-2 cells, we provide evidence that localized adherence and diffuse adherence by EPEC are due to at least two genetically distinct adhesions which confer phenotypic differences in both the morphology of HEp-2 cell adherence and in surface hydrophobicity. The two factors are each encoded on plasmids which vary in size from 55 to 70 megadaltons; one strain exhibiting localized adherence carried these genes on the chromosome.