Objective. To test the hypothesis that methicillin-susceptible Staphylococcus aureus (MSSA) carriage may protect against nosocomial methicillin-resistant S. aureus (MRSA) acquisition by competing for colonization of the anterior nares. Design. Prospective cohort and nested case-control study. Setting. Swiss university hospital. Patients. All adult patients admitted to 14 wards of the general medicine division between April 1 and October 31, 2007. Methods. Patients were screened for MRSA and MSSA carriage at admission to and discharge from the division. Associations between nosocomial MRSA acquisition and MSSA colonization at admission and other confounders were analyzed by univariable and multivariable analysis. Results. Of 898 patients included, 183 (20%) were treated with antibiotics. Nosocomial MRSA acquisition occurred in 70 (8%) of the patients (case patients); 828 (92%) of the patients (control subjects) were free of MRSA colonization at discharge. MSSA carriage at admission was 20% and 21% for case patients and control subjects, respectively. After adjustment by multivariate logistic regression, no association was observed between MSSA colonization at admission and nosocomial MRSA acquisition (adjusted odds ratio [aOR], 1.2 [95% confidence interval (CI), 0.6-2.3]). By contrast, 4 independent predictors of nosocomial MRSA acquisition were identified: older age (aOR per 1-year increment, 1.05 [95% CI, 1.02-1.08]); increased length of stay (aOR per 1-day increment, 1.05 [95% CI, 1.02-1.09]); increased nursing workload index (aOR per 1-point increment, 1.02 [95% CI, 1.01-1.04]); and previous treatment with macrolides (aOR, 5.6 [95% CI, 1.8-17.7]). Conclusions. Endogenous MSSA colonization does not appear to protect against nosocomial MRSA acquisition in a population of medical patients without frequent antibiotic exposure.