We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of β-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-β-D-galactoside, an inducer of β-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for β-galactosidase assay in wells. The strains used differed widely in their β-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10⁵ bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10⁷ bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.