A major reservoir of Escherichia coli O157:H7 is the intestine of healthy cattle, however, little is known about the ecology and dissemination of this human pathogen. The aim of this study was to determine the prevalence and to characterize E. coli O157:H7 in house flies from a beef cattle farm over a five-month period. Fifty adult house flies were collected (four times per week) from June till October 2003 from two sites on a cattle feedlot in Kansas. Individual flies (n=3440) were homogenized in PBS buffer, serially diluted, and plated onto sorbitol MacConkey agar with cefixime and tellurite using a direct drop plate technique. Sorbitol-negative colonies were screened for O157 serogroup by the latex agglutination test, positive colonies were counted, isolated, and identified by the API Rapid 20E test. E. coli O157 isolates were tested for the virulence genes (stx 1, stx 2, eaeA) and flagellar H7 gene (fliC) by PCR. Resistance (MIC) of isolates to ten antibiotics was determined. Genotypic diversity of E. coli O157:H7 isolates was assessed by PFGE. Number of fecal coliforms in individual flies were determined using mFC agar. Our results will presented and discussed. The data indicate that house flies are an important vector of E. coli O157:H7. House flies carry virulent E. coli O157:H7 as well as a large community of fecal coliforms. The high populations of house flies around cattle present a potential mechanism for the dissemination of E. coli O157:H7.