Human immunodeficiency virus (HIV) is mainly transmitted via sexual activity, mother-to-child transmission, and contact with body fluids, such as saliva and semen. Cerumen, however, has not been investigated for its capability to transmit HIV. The purpose of this study was to evaluate the potential of cerumen for transmission of HIV infection. This study was conducted among 42 treatment-naive HIV-infected patients with positive HIV RNA and 27 HIV-infected patients with negative HIV RNA receiving antiviral treatment. Simultaneous blood samples were studied as positive controls. Sixty-nine prospectively collected cerumen specimens were analyzed for the presence of HIV RNA by real-time polymerase chain reaction (PCR). None of the 69 cerumen specimens were positive for HIV RNA. These results conclude that cerumen in HIV-positive patients with or without antiretroviral therapy (ART) carry only an insignificant risk of transmission. However, standard infection control precautions should be applied carefully in all examinations and surgical operations of the ears.