A cell-free system derived from Escherichia coli has been used to study the kinetics of inhibition of peptide bond formation by spermine at optimal Mg2+ concentration (10 mM). With the aid of the puromycin reaction, it was possible to show that spermine does not affect the final degree of peptide bond formation. However, spermine inhibits peptide bond formation at the kinetic phase of the reaction. A single molecule of spermine participates in the mechanism of inhibition. The type of inhibition of peptide bond formation by spermine is simple competitive, regardless of whether the ternary complex AcPhe-tRNA-poly(U)-ribosome (complex C) is formed in the presence (Ki = 190 microM) or in the absence (Ki = 84 microM) of factors washable from ribosomes. Preincubation experiments of spermine with the individual components of complex C demonstrated that the inhibitory effect of spermine is closely related with its binding to AcPhe-tRNA.