Ambulances are frequently contaminated with infectious microorganisms shed by patients during transport that can be transferred to subsequent patients and emergency medical service workers. Manual decontamination is tedious and time-consuming, and persistent contamination is common even after cleaning. Ultraviolet germicidal irradiation (UVGI) has been proposed as a terminal disinfection method for ambulance patient compartments. However, no published studies have tested the use of UVGI in ambulances. The objectives of this study were to investigate the efficacy of a UVGI system in an ambulance patient compartment and to examine the impact of UVGI fixture position and the UV reflectivity of interior surfaces on the time required for disinfection. A UVGI fixture was placed in the front, middle, or back of an ambulance patient compartment, and the UV irradiance was measured at 49 locations. Aluminum sheets and UV-reflective paint were added to examine the effects of increasing surface reflectivity on disinfection time. Disinfection tests were conducted using Bacillus subtilis spores as a surrogate for pathogens. Our results showed that the UV irradiance varied considerably depending upon the surface location. For example, with the UVGI fixture in the back position and without the addition of UV-reflective surfaces, the most irradiated location received a dose of UVGI sufficient for disinfection in 16 s, but the least irradiated location required 15 hr. Because the overall time required to disinfect all of the interior surfaces is determined by the time required to disinfect the surfaces receiving the lowest irradiation levels, the patient compartment disinfection times for different UVGI configurations ranged from 16.5 hr to 59 min depending upon the UVGI fixture position and the interior surface reflectivity. These results indicate that UVGI systems can reduce microbial surface contamination in ambulance compartments, but the systems must be rigorously validated before deployment. Optimizing the UVGI fixture position and increasing the UV reflectivity of the interior surfaces can substantially improve the performance of a UVGI system and reduce the time required for disinfection.