The glycylcyclines are new tetracycline derivatives that include the N,N-dimethylglycylamido derivative of minocycline (DMG-MINO) and the N,N-dimethylglycylamido derivative of 6-demethyl-6-deoxytetracycline (DMG-DMDOT). The susceptibilities of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum to DMG-MINO, DMG-DMDOT, tetracycline, doxycycline, and minocycline were determined by the agar dilution method. The glycylcyclines with MICs at which 50% of the isolates are inhibited of 0.25 to 0.5 mu g/ml for M. pneumoniae were two- to fourfold more active than tetracycline and had the same activity as minocycline and doxycycline. Tetracycline-susceptible M. hominis strains were four- to eightfold more susceptible to the glycylcyclines (0.12 to 0.25 mu g/ml) than to tetracycline. Strains of M. hominis known to be resistant to tetracycline, doxycycline, and minocycline because of the tet(M) determinant were as susceptible to the glycylcyclines as the tetracycline-susceptible strains. For tetracycline-susceptible U. urealyticum strains, the glycylcyclines showed the same activity as tetracycline (MICs at which 50% of the isolates are inhibited of 1 to 2 mu g/ml). Tetracycline-resistant strains of U. urealyticum were resistant to doxycycline and minocycline and showed variable susceptibility to the glycylcyclines (range, 0.5 to 32 mu g/ml). In view of the increasing resistance of M. hominis and U. urealyticum strains to tetracyclines, the glycylcyclines have promise, pending assessment of their pharmacokinetic and safety profiles,