OBJECTIVE: Risk factors for rectal carriage of ESBL-E and transmission were investigated in an outbreak of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E). DESIGN: Rectal carriage of ESBL-E was determined in a cross-sectional survey by culture of perianal swabs or fecal samples. Both phenotypical and genotypical methods were used to detect the production of ESBL. Nosocomial transmission was defined as the presence of genotypically related strains in ≥2 residents within the NH. Patient characteristics and variables in infection control practices were registered to investigate risk factors for transmission. SETTING: A nursing home (NH) in the southern Netherlands. PARTICIPANTS: Of 189 residents, 160 residents (84.7%) were screened for ESBL-E carriage. Of these 160 residents, 33 (20.6%) were ESBL-E positive. ESBL carriage rates varied substantially between wards (range, 0-47%). Four different ESBL-E clusters were observed. A bla CTX-M1-15 positive E. coli ST131 constituted the largest cluster (n=21) and was found in multiple wards (n=7). RESULTS: Our investigation revealed extensive clonal dissemination of bla CTX-M1-15-positive E. coli ST131 in a nursing home. Unexplained differences in ESBL prevalence were detected among the wards. CONCLUSIONS: As NHs constitute potential sources of multidrug-resistant bacteria, it is important to gain a better understanding of the risks factors and routes of transmission of ESBL-E.