An in vitro model and an image analysis were designed to improve on existing quantification methods in the assessment of the adherence of Enterobacteriaceae to human epithelial cell monolayers. Adherence to HeLa cell monolayers of three oral isolates and one type strain from each of four species of Enterobacteriaceae over two incubation time periods was examined. Correction for actual cell area and a cube root transformation of the data to stabilize variance were applied. While behaviour varied between strains within species, E. cloacae was the most, and K. pneumoniae the least, adherent species irrespective of the incubation period. Increasing the incubation period from 30 min to 60 min resulted in greater adherence for E. cloacae, E. coli, and C. freundii, but not K. pneumoniae strains. The method permits the reliable measurement and valid analysis of the adherence of Enterobacteriaceae to cultured epithelial cell monolayers.