Background: Colonization pressure has been identified as an important risk factor in the transmission of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus species, but the role of colonization pressure in the transmission of Clostridium difficile-associated disease (CDAD) is unclear. The purpose of this study was to evaluate CDAD pressure, a modified form of colonization pressure based on symptomatic CDAD cases, as a risk factor for CDAD. Methods: Retrospective cohort and nested case-control studies of patients admitted to Barnes-Jewish Hospital from January 1, 2003, through December 31, 2003. Univariate analysis and multivariate logistic regression models were used to evaluate the role of CDAD pressure as a risk factor for CDAD. Results: A total of 36 275 patients were included in the cohort, of which 382 had CDAD. The median CDAD pressure was higher for case patients than noncase patients (1.4 vs 0.3; P<.001), and only 1 patient with CDAD had a CDAD pressure of 0. In the nested case-control study, CDAD pressure remained an independent risk factor for CDAD after adjustment for demographics, severity of illness, medications received (chemotherapy, gastric acid suppressors, antidiarrheals or narcotics, and antibiotics), and abdominal procedures or surgery performed. Conclusions: The results of this study suggest thatCDAD pressure may be an independent risk factor for CDAD. Future studies that evaluate risk of CDAD should control for CDAD pressure.