Isolates of Pasteurella testudinis recovered from clinically healthy desert tortoises (Gopherus agassizii) and tortoises with upper respiratory tract disease (URTD) were characterized in an attempt to identify strains associated with disease. Eighty-nine isolates, 52 from ill and 37 from healthy tortoises collected from Nevada (USA), June 1990 to September 1991, were genomically fingerprinted and grouped based on ribotype similarity. Twelve isolates (six from ill and six from healthy tortoises) were further characterized with regard to whole-cell protein (WCP) and outer membrane protein (OMP) composition and their ability to survive in normal tortoise plasma. The 89 isolates were initially distributed into 33 distinct ribotype groups using the restriction enzyme EcoRI; five ribotypes contained over 50% of the isolates. Only one EcoRI ribotype was comprised of multiple isolates (n = 4) exclusively recovered from tortoises with URTD. When the ten EcoRI ribotypes that contained more than one isolate per ribotype were further studied using a second restriction enzyme, EcoRV, one EcoRI/EcoRV ribotype contained five isolates recovered from URTD tortoises and none from healthy animals. The EcoRI ribotype comprised of four isolates, all from tortoises with URTD, was further separated into three distinct groups with EcoRV. All 12 isolates studied grew equally well in normal tortoise plasma, and when broth-grown WCP and OMP profiles were evaluated, no proteins were unique to isolates from URTD tortoises. Iron-regulated OMP's were produced in three isolates examined, but these OMP's apparently were not virulence-related.