The in vitro suscetibilities of six commonly used antimicrobial agents against 29 isolates of intestinal spirochetes isolated from dogs in Japan were examined by the agar dilution technique. In addition, the genetic basis of tylosin resistance in in vitro selected resistant mutants of two reference strains and three tylosin-susceptible field isolates obtained by three successive subcultures on blood agar containing 1 μg/ml of tylosin was investigated. Carbadox was the most active (MIC: < 0.00625) of all the antimicrobial agents. Although all the isolates were susceptible to tylosin, some were resistant to erythromycin. Tiamulin, lincomycin and dimetridazole were also very active against the isolates. All the resistant isolates did not harbor any plasmids. In vitro selected tylosin-resistant mutants of previously tylosin-susceptible isolates showed a new mutation in which their adenine at the base position equivalent to 2,062 of 23S rDNA of Escherichia coli has been replaced by cytosine. These findings may both provide guidance towards the proper choice of antimicrobial agents for the treatment of canine intestinal spirochetosis, and add to the understanding of the genetic basis of tylosin resistance.