The two peptide-like antibiotics ficellomycin and feldamycin impair semiconservative DNA replication but not DNA repair synthesis in bacteria. Specifically both antibiotics cause the accumulation of a 34S DNA species in toluenized Escherichia coli cells which lacks the capability of being integrated into larger DNA pieces and eventually the complete bacterial chromosome. Novobiocin, a known inhibitor of replicative DNA synthesis, was investigated for comparative purposes. The action of this latter antibiotic differs from the ones exerted by ficellomycin and feldamycin in the novobiocin appears to block an event associated with the initiation of Okazaki fragments. The fact that novobiocin impairs DNA gyrase suggests that this enzyme plays an essential role during the initiation of Okazaki pieces.