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When cultures of biotin-requiring mutant of Brevibacterium divaricutum NRRL-2311 were treated in early logarithmic phase with penicillin (2 -3 units/ml) in a glucose-mineral medium under excess supply of biotin, large amounts of a high-molecular-weight material accumulated in the medium. The phenomenon could not be observed with cultures run in parallel from which penicillin was omitted. Chemical analyses of the excreted material isolated from the media of I-h and 24-h penicillin-treated cultures, showed that the main constituents were peptidoglycan components of noncross-linked structure bearing both L-and D-alanine residues ; evidence was also obtained for the occurrence of extractable lipid material, non-amino sugars and organic phosphate.Under identical conditions, the excretion of peptidoglycan could be induced by ampicillin and cloxacillin, respectively, but not by bacitracin. Addition of penicillin to biotin-requiring mutant of M . glutamicus yielded similar results, indicating that the phenomenon was not restricted to the Brev. divaricutum strain. This suggests that excretion of peptidoglycan material by the two biotin-requiring mutants might be the result of two events, (a) a change in the osmotic barrier of the cell and (b) specific inhibition of cross-link formation in peptidoglycan induced by penicillin.Several procedures were examined for the isolation and purification of the peptidoglycan complex excreted by penicillin-treated Brev. mutant. Suitable labelling experiments with L-[U-14C]glutamic acid and analyses of lysozyme digests of the purified fractions, suggest that some of the components might contain murein fragments covalently linked to a polysaccharide portion.During our studies on the accumulation of amino acids into the culture broth of the biotin-requiring mutant of Brevibacteriurn divaricatum (NRRL-231 l ) , it has been observed that in the presence of low doses of penicillin the extracellular accumulation of glutamate is accompanied by the excretion of some highmolecular-weight material. The first observations were made in cultures grown on glucose media containing biotin, to which penicillin G (2-10 units/ml) was added at the early logarithmic phase; incubations were carried out at 28 "C for 24, 48 and 72 h, respectively.When a growing culture was incubated simultaneously with 14C-labelled L-glutamic acid and penicillin for 24 h and the supernatant fluid submitted to gel fractionation, a substantial portion of radioactivity eluted a t low retention volumes. I n contrast, in the culture fluid of the control from which penicillin had been omitted, no radioactivity associated with some high-molecular-weight material could be detected. When subjected to paper electrophoresis and chromatography, the radioactive material excreted by penicillin-treated cells did not move from the origin. Hydrolysis of a sample in 6 M HCl a t 100 "C revealed diaminopimelic acid as one of the amino acid components.Since diaminopimelate is present in no other cellular material than in murein, the excreted mater...