Felid herpesvirus 1 (FeHV-1) is an important feline pathogen of the upper respiratory tract which can be identified in clinical cases by virus isolation and PCR. Studies on the effect and mode of spread of FeHV-1 in the respiratory epithelium, however, have previously only been performed in infected cats. Feline tracheal organ cultures which were inoculated with FeHV-1 at varying multiplicity of infection (MOI) were established. A dose-dependent response was observed. Low MOIs induced multifocal infection in the otherwise viable respiratory epithelium, which allowed monitoring of viral growth over several days. Therefore, tracheal organ cultures represent a suitable model for further study of the morphological and functional effects of FeHV-1 on respiratory epithelium, mimicking the in vivo situation.