A simple procedure for obtainig drug-resistant strains of Acholeplasma, occuring naturally and spontaneously at a low frequency in a broth culture was described. The drug-resistant strains could be obtained and enriched for by the addition of more than twice volumes of fresh broth containing antibiotics to a pre-cultured medium with Acholeplasma at the plateau phase of the growth. Furthermore, the growth of drug-resistant cells was promoted by the addition of broth containing protease and an antibiotics. By means of this procedure, various kinds of antibiotics-resistant strains of Acholeplasma were easily isolated, some of which were not obtained by the agar plate selection method. The merits of this procedure were that the scale of this system could be controlled and that the isolation of drug-resistant strain was very simple and rapid.