Cells of Proteus mirabilis, synchronized by sucrose density gradient centrifugation, were grown in complex medium containing radioactive N-acetylglucosamine. At various times, labelled murein sacculi were isolated and digested with endo-N,O-acetylmuramidase from Chalaropsis. The murein fragments thus obtained were separated into disaccharide peptides as the monomeric subunits and into peptide-cross-linked subunits by gel filtration. The subunits were further differentiated into O-acetylated and non-O-acetylated species, and into subunits containing anhydro-N-acetylmuramic acid which were glycan chain terminators in the native sacculi. Quantification of the subunit species gave the following results. At specific times during the cell cycle, murein subunits were lost from the polymer and a transient decrease in cross-linkage was observed. The overall degree of cross-linkage in mature murein, i.e. the ratio of peptide-cross-linked subunits versus uncross-linked subunits, was 1.15 as determined by regression analysis. Anhydro-N-acetylmuramic-acid-containing murein subunits representing glycan chain terminators were found either peptide-cross-linked or uncross-linked as monomers. Since these two subunit species were recovered in a defined ratio of 1.6, mature murein consisted of at least two different types of glycan chains. On average, each chain contained 15.4 murein subunits. About 60% of the murein subunits in mature murein were O-acetylated and showed a higher degree of cross-linkage than the non-O-acetylated portion. Finally, following the composition of the sacculus during the cell cycle revealed a complex precursor-product relationship between non-O-acetylated and O-acetylated subunits during murein maturation. The data allowed us to deduce several features of the assembly process of murein sacculi.