OBJECTIVE. To determine the prevalence of asymptomatic carriage of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) among healthcare professionals (HCPs) who experience varying degrees of exposure to ambulatory patients and to genetically characterize isolates. METHODS. This single-center, cross-sectional study enrolled 256 staff from the intensive care units, emergency department, and prehospital services of an urban tertiary care university hospital in 2008. Occupational histories and nasal samples for S. aureus cultures were obtained. S. aureus isolates were genetically characterized with the use of spa typing and screened for mecA. MRSA isolates underwent further characterization. RESULTS. S. aureus was isolated from 112 of 256 (43.8%) HCPs, including 30 of 52 (57.7%) paramedics, 51 of 124 (41.1%) nurses, 11 of 28 (39.3%) clerical workers, and 20 of 52 (38.5%) physicians. MRSA was isolated from 17 (6.6%) HCPs, including 1 (1.9%) paramedic, 13 (10.5%) nurses, 1 (3.6%) clerical worker, and 2 (3.8%) physicians. Among S. aureus isolates, 15.2% were MRSA. MRSA prevalence was 9.6% (12/125) in emergency department workers, 5.1% (4/79) in intensive care unit workers, and 1.9% (1/52) in emergency medical services workers. Compared with paramedics, who had the lowest prevalence of methicillin resistance among S. aureus isolates (1 of 30 [3.3%] isolates), nurses, who had the highest prevalence (13 of 51 [25.4%] isolates), had an odds ratio of 9.92 (95% confidence interval, 1.32-435.86; P = .02) for methicillin resistance. Analysis of 15 MRSA isolates revealed 7 USA100 strains, 6 USA300 strains, 1 USA800 strain, and 1 EMRSA-15 strain. All USA300 strains were isolated from emergency department personnel. CONCLUSIONS. The observed prevalence of S. aureus and MRSA colonization among HCPs exceeds previously reported prevalences in the general population. The proportion of community-associated MRSA among all MRSA in this colonized HCP cohort reflects the distribution of the USA300 community-associated strain observed increasingly among US hospitalized patients. © 2010 by The Society for Healthcare Epidemiology of America. All rights reserved.