The MICs of various macrolide, lincosamide and streptogramin B antibiotics against highly erythromycin-resistant Escherichia coli 209K strain were evaluated. E. coli 209K showed high MICs against 14-membered macrolides and the relatively weaker resistance to 16-membered macrolides, lincosamides and streptogramin B. The macrolide-phosphotransferase K from E. coli 209K showed greater substrate specificity to the 14-membered macrolide antibiotics than to the 16-membered macrolide antibiotics, lincosamide and streptogramin B. Therefore, it was considered that the high resistance was due to the macrolide-phosphotransferase K.