Steffimycin interferes with amino acid incorporation mediated by synthetic polyribonucleotides in cell-free bacterial systems. This inhibition is a secondary activity of the antibiotic, which acts primarily as a suppressor of RNA synthesis in bacteria and bacterial cell-free systems. Inhibition of peptide biosynthesis is apparent only at a drug concentration higher than that necessary for inhibition of RNA synthesis in cell-free systems. The chelating properties of the antibiotic toward divalent cations do not appear to be responsible for inhibition of peptide biosynthesis. Reversal studies suggested that excess transfer RNA but not 30S fraction or polyuridylic acid reduces steffimycin inhibition of amino acid incorporation. Difference spectra obtained from antibiotic-transfer RNA mixtures indicate that steffimycin binds to transfer RNA. It is concluded that the binding of steffimycin to transfer RNA is the basis for its interference with amino acid incorporation. © 1969.