Two assays were developed to quantitate the adherence of an Escherichia coli strain (RDEC-1) known to colonize the mucosal surface of the small intestine of rabbits to brush borders isolated from rabbit intestinal epithelial cells. In the first assay, the mean adherence per rabbit brush border was determined by counting the number of organisms adhering to each of 40 brush borders under phase microscopy. The mean adherence of RDEC-1 (11.5 ± 0.7 per rabbit brush border) was significantly greater than adherence of two nonpathogenic strains: HS (2.7 ± 0.4 per rabbit brush border) and 640 (0.8 ± 0.1 per rabbit brush border). A similar distinction between the adherence of RDEC-1 and the control (nonadherent) organisms could be made more rapidly by determining the percentage of the total number of brush borders which had 10 or more adherent organisms; this second assay was used to define the optimum conditions for adherence. Maximum adherence was seen within 15 min. Adherence was temperature dependent, with adherence after 1 min at 37°C being fourfold greater than that at 4°C. The pH optimum for adherence was between 6.5 and 7.0, and adherence was abolished below pH 5.0. With the first, more sensitive assay, the effect of electrolytes and a number of hexoses and hexosamines on adherence was analyzed. RDEC-1 adherence was inhibited at high ionic strengths; however, adherence was not influenced at moderately high concentrations (20 mg/ml) by either d-mannose or l-fucose, in contrast to the case for other reported enteric pathogens. These two quantitative in vitro assays for adherence produce consistent results and have been used to partially characterize the adherence of RDEC-1 to rabbit brush borders.