When using tissue culture as a tool for screening antitumor agents, most cell cultures were contaminated with mycoplasma and attempts were made to eliminate the mycoplasma from the cell cultures with antibiotics. Several strains of mycoplasma tested were highly sensitive to macrolide antibiotics and antitumor antibiotics. Among macrolide antibiotics, leucomycin group such as leucomycin, spiramycin and tylosin were more active than erythromycin group, such as erythromycin and oleandomycin. In contrast to the results in vitro, mycoplasma infected naturally in the cultured cells was highly resistant to the antibiotics, but mycoplasma (strain Campo) inoculated in embryonated eggs was completely eliminated with 2 injections of 40mcg/egg of leucomycin.