The composition and the mode of insertion of peptidoglycan synthesized during the cell cycle of Escherichia coli were determined. This was carried out on peptidoglycan that was periodically pulse-labeled in synchronously growing cultures. The chemical composition of the pulse-labeled (newly synthesized) peptidoglycan remained constant throughout the cell cycle, as judged from high-pressure liquid chromatography analysis of the muropeptide composition. The mode of insertion was deduced from the acceptor-donor radioactivity ratio in the bis-disaccharide tetratetra compound. The ratio was low in elongating cells and high in constricting cells. This indicates that during elongation, peptidoglycan was inserted as single strands, whereas during constriction, a multistranded (or sequential single-stranded) insertion occurred. Experiments with an ftsA division mutant suggested that the composition and mode of insertion of newly synthesized peptidoglycan remained the same throughout the constriction process. Our results imply that the changed mode of insertion rather than the chemical structure of the peptidoglycan might be responsible for the transition from cell elongation to polar cap formation.