Staphylococcal gingival flora was characterized in cultures from 135 dogs. Staphylococcus intermedius was isolated in 39% of the cultures, S. aureus was isolated in 10%, and both were isolated in 2.0%. S. aureus was isolated more often from dogs of working breeds with weights of greater than 40 lb (ca. 18 kg) and with outdoor habitats than was S. intermedius, which was associated with dogs of nonworking breeds with weights of less than 40 lb and indoor habitats. S. intermedius was distinguished from S. aureus by the following characteristics: coagulation of rabbit plasma at 4 h (26 versus 100%, respectively), hemolysis of sheep blood at 24 h (30 versus 79%, respectively), and mannitol fermentation at 24 h (4 versus 93%, respectively). A clear separation of the two species was apparent only with the acetoin (modified Voges-Proskauer) reaction (100% of the S. aureus isolates versus 0% of the S. intermedius isolates) and beta-galactosidase activity on the API Staph-Ident strip (0% of the S. aureus isolates and 100% of the S. intermedius isolates). Susceptibilities of S. intermedius and S. aureus were 72 and 7%, respectively, to penicillin G, and 100% of both species to oxacillin. Fourteen previously collected strains of coagulase-positive staphylococci from infected canine-inflicted human wounds were reanalyzed; 3 of 14 (21%) isolates were S. intermedius. We conclude that S. intermedius is a common canine gingival flora and is responsible for some canine-inflicted human wound infections, thus representing a newly recognized zoonotic pathogen.