C. difficile-associated diarrhea (CDAD) has become a major cause of morbidity in hospitalized patients. In this study of five-year (2001-2005, inclusive) trends of incidence of CDAD among adults in an inner-city medical center, the overall annual incidence increased from 5.08 to 8.42 cases/10(3) admissions (p = 0.0005). Age distribution remained fairly constant for 2001-2004 but decreased significantly in 2005 (p = 0.005); no significant change was observed for gender. During the five-year period, we observed a decline in the use of histamine type 2 receptor antagonists (H2A) with a concomitant increase in the use of proton pump inhibitors (PPI) as a prophylactic measure to prevent stress ulcers. The usage of PPI correlated exactly (r(s) = 1.0; p = 0.017) with the increase in CDAD incidence. A case (n = 122)-control (n = 244) study for the final year was conducted, examining the association of PPI and H2A with CDAD. After controlling for the effect of antibiotic use, PPI either pre- or during admission was associated with CDAD (odds ratio, OR (adjusted) = 2.75, 95% CI = 1.68 to 4.52; p = 0.0001); the association with H2A was not significant (OR (adjusted) = 0.95, 95% CI = 0.39 to 2.34; p = 0.9153). If only first-time use during hospital stay is considered, PPI were also strongly associated with CDAD (OR (adjusted) = 1.88, 95% CI: 1.07 to 3.31; p = 0.0283) and H2A were not associated with CDAD (OR (adjusted) = 0.73, 95%CI: 0.26 to 2.06; p = 0.5520). These data suggest that the widespread prescription of PPI for stress ulcer prophylaxis in acute care facilities may contribute to the increased incidence of CDAD.