Lysozyme in combination with ethylenediamine tetraacetic acid (EDTA) gives rise to spheroplasts whenMycobacterium tuberculosisis cultured in a nutritionally complete Tween 80 – albumin medium. These spheroplasts do not multiply in this medium, but when such forms, stabilized by the presence of sucrose and Mg++, are inoculated into PPLO agar or broth an L-form of growth is initiated. A CO2requirement was demonstrated by L-forms grown on solid media.