OBJECTIVE: To perform an initial screening study of methicillin-resistant Staphylococcus aureus (MRSA) contamination in an ambulance fleet. METHODS: This was a cross-sectional study of MRSA contamination in an ambulance fleet operating in the western United States in June 2006. Five specific areas within each of 21 ambulances (n = 105) were tested for MRSA contamination using dacron swabs moistened with a 0.85% sterile saline solution. These samples were then plated onto a screening media of mannitol salt agar containing 6.5% NaCl and 4 mcg/mL oxacillin. RESULTS: Thirteen samples isolated from 10 of the 21 ambulances (47.6%) in the sample group tested positive for MRSA. CONCLUSIONS: The results of this preliminary study suggest that ambulances operating in the emergency medical services (EMS) system may have a significant degree of MRSA contamination and may represent an important reservoir in the transmission of potentially serious infections to patients.