Mycoplasma pneumoniae was made highly resistant to erythromycin in vitro by serial subculture in broth media containing erythromycin. The resistance developed to erythromycin was 200 mug/ml with the Mac strain, a prototype of M. pneumoniae, and 10 mug/ml with the Fukumura strain, an isolate. The erythromycin resistance was accompanied by cross resistance to other macrolide antibiotics (leucomycin, josamycin, spiramycin, and oleandomycin) and to lincomycin, but there was no resistance to vernamycin B. Resistance to the antibiotics developed in vitro or in vivo was stable after the microorganisms were repeatedly transferred in antibiotic-free media.