A temperature-sensitive (Ts) mutant strain of Ornithobacterium rhinotracheale (ORT) was developed after exposure of the wild-type organism to N-methyl-N′-nitro-N-nitrosoguanidine. The Ts mutant strain grew at 31 C but had its growth inhibited at 41 C unlike wild-type parent strain. The Ts mutant and parent strains were characterized. Morphologic and biochemical properties of wild-type and mutant strains did not show any differences. The strains were also characterized by polymerase chain reaction (PCR)-based fingerprinting methods. Results showed similar patterns in repetitive sequences by repetitive PCR (enterobacterial repetitive intergenic consensus, highly conserved repeated DNA elements present in Streptococcus pneumoniae (BOX), repetitive extragenic palindromic, and Salmonella enteritidis repetitive element primers); however, random amplified polymorphic DNA fingerprinting was able to differentiate mutant and parent strains showing a unique pattern for each of the ORT strains. The rationale for the use of a Ts strain as a vaccine is based on the ability of the mutant to colonize the upper respiratory tract but not the lower respiratory tract and systemic system of the birds, where the wild-type strain causes severe lesions. In a preliminary evaluation, Ts strain of ORT was recovered from tracheas and choanae of Ts-treated turkeys for 13 days postadministration of the strain either in drinking water or by oculonasal instillation. Humoral immune response was detected in Ts-vaccinated but not in control group birds after 3 wk postadministration. Results suggest that Ts strain of ORT has promising potential use as a live vaccine for ORT.