Abstract Clostridium difficile infection (CDI) is the main identifiable cause of antibiotic assoicated diarreah. The incidence of CDI is approximately 677/100,000 patients resulting in close to 500,000 cases annually in the US alone. CDI causes approximately 25,000 deaths a year and costs the health care system an estimated $6.3 billion/year. The main CDI risk factor is aggressive broad-spectrum antibiotic use. Similarly, older populations are more susceptible to CDI than younger cohorts. Conditions that reduce immunity (e.g. organ transplant, chemotherapy, AIDS) are also strongly correlated with CDI severity. Importantly, studies have shown that women have higher risk of CDI than men. CDI symptoms range from asymptomatic colonization to mild diarrhea to deathly colitis. All studies that delineate risk factors associated with CDI have been conducted with infected populations. Hence, the determinants of individual predisposition to contracting CDI are not well understood. Similarly, it is not clear why CDI symptoms severity vary among individual patients. Mice have been used successfully as a model to test novel approaches to treat for C. difficile infections. The murine CDI model present symptoms progression reminiscent of human CDI and respond to the same treatments. During our screening for CDI prophylactics, we observed that female mice developed more severe CDI signs compared to their male counterpart. In this application, we will test the hypothesis that sex hormone levels correlate with CDI symptom onset and severity. We will also determine whether sex-related variables affect CDI prevention and treatment. The data obtained in this project will allow determining the contribution of steroidal sex hormones and/or sexual status to murine CDI susceptibility. This information will then be used in a follow-up R01 application to determine the mechanisms underlying the differences between male and female CDI susceptibility.