Sensitivity of spheroplasts (prepared in two ways) of a colicin-sensitive strain, of colicin-resistant and of colicin-tolerant mutants and of strains immune to colicins E1 and E2 was estimated and compared. Generally, the removal of the peptidoglycan layer brought about a slight nonspecific support for colicin translocation across the cell wall in sensitive, tolB tolerant and immune bacteria. tolB spheroplasts were colicin E1-sensitive, but E2-insensitive. Spheroplasts were always fragile and lysed spontaneously, especially those produced by lysozyme. Bacteria carrying tolA, tolQ and tolR mutations kept their colicin insensitivity as spheroplasts, just as the resistant ones. Bacteria rendered colicinogenic and hence colicin-immune turned to high colicin sensitivity in spheroplast form. The results indicate a change in plasma membrane associated with the spheroplast formation.