The in vitro synergistic effects of combinations of EDTA-tromethamine and five antimicrobial agents (ampicillin, cephalexin, oxytetracycline, streptomycin and sulphadimethoxine) on three clinically isolated Gram-positive bacteria (Staphylococcus aureus, Staphylococcus hominis and Streptococcus faecium) were investigated. The bacteria had been isolated from three cases of canine otitis resistant to beta-lactam antibiotic therapy. The antimicrobial activity was evaluated by measuring the minimal inhibitory concentration for the antibiotics alone or in combination with EDTA-tromethamine. EDTA-tromethamine potentiated the activity of cefalexin against S. aureus and S. hominis, of oxytetracycline against S. aureus and S. faecium and of streptomycin against S. faecium. No significant effects were noted on the activity of oxytetracycline against S. hominis. The remaining combinations gave a slight synergistic effect. As previously shown for Gram-negative resistant bacteria, these data suggest that the association of EDTA-tromethamine and appropriate antibiotic therapy may be useful to overcome persistent infections of soft tissues in domestic animals.