Thirty eight-week-old Beagle puppies were randomly divided into 3 groups which consisted of 10 puppies that received a single intranasal kennel cough vaccine 48 h before challenge with B. bronchiseptica (Group I), 10 puppies that were vaccinated 72 h before challenge (Group II) and 10 puppies that served as unvaccinated controls (Group III). Body temperature was measured in all groups daily starting on the day of challenge until 14 days. It was shown that control Group III developed clinical signs of infectious tracheobronchitis after challenge, including pyrexia, spontaneous or induced coughing, clear or mucopurulent nasal or ocular discharge, nasal congestion and dry or wet rales upon ausculation. B. bronchiseptica was isolated from all animals in this group. Group I puppies had similar but less severe clinical signs, while Group II had almost no clinical signs, except for 1 puppy. The average total Mann-Whitney U score for clinical signs in Group II was significantly lower compared to Group III, while no significant difference was found between Groups I and III. These results show that the earliest onset of immunity achievable for the B. bronchiseptica fraction of the modified live bivalent vaccine is between 48 and 72 h after intranasal administration.