In 2002, a Scottish bat conservationist developed a rabies-like disease and subsequently died. This was caused by infection with European bat lyssavirus 2 (EBLV-2), a virus closely related to Rabies virus (RABV). The source of this infection and the means of transmission have not yet been confirmed. In this study, the hypothesis that lyssaviruses, particularly RABV and the bat variant EBLV-2, might be transmitted via the airborne route was tested. Mice were challenged via direct introduction of lyssavirus into the nasal passages. Two hours after intranasal challenge with a mouse-adapted strain of RABV (Challenge Virus Standard), viral RNA was detectable in the tongue, lungs and stomach. All of the mice challenged by direct intranasal inoculation developed disease signs by 7 days post-infection. Two out of five mice challenged by direct intranasal inoculation of EBLV-2 developed disease between 16 and 19 days post-infection. In addition, a simple apparatus was evaluated in which mice could be exposed experimentally to infectious doses of lyssavirus from an aerosol. Using this approach, mice challenged with RABV, but not those challenged with EBLV-2, were highly susceptible to infection by inhalation. These data support the hypothesis that lyssaviruses, and RABV in particular, can be spread by airborne transmission in a dose-dependent manner. This could present a particular hazard to personnel exposed to aerosols of infectious RABV following accidental release in a laboratory environment.