AbstractVancomycin, an inhibitor of peptidoglycan synthesis, depressed the growth of Mycobacterium sp. NRRL B 3805 and MB 3683, but not the β‐sitosterol side chain degradation and androstane derivatives accumulation. As a result, the specific activity (products formed/cell weight unit × h) increased threefold in the peptidoglycan‐deficient cells, indicating faster crossing of the sterol through the cell water barrier.Cell wall preparations: crude cell wall (CCW), purified cell wall (PCW), and peptidoglycan — enriched PCW — residue (PEPCW) were obtained and analysed in order to find a relationship between the vancomycin — induced chemical changes and the permeation rate of the sterol.The amounts of CCW, PCW and PEPCW, produced from 8 g lyophilised control cells were 445, 170 and 28 mg respectively. The respective figures were 176, 61, and 4.8 mg for vancomycin — treated cells. In addition to the lower content of the rigid layer, a distinct shift in the molar ratios of the peptidoglycan constituents: alanine, glutamic, diaminopimelic and muramic acids, and glucosamine was observed under the action of the murein inhibitor. The most significant change was that of muramic acid: diaminopimelic acid molar ratio, the compounds which are markers of glycan strands and tetrapeptides, respectively. In control cells it was approximately 1:1, and increased to 1.34–1.43: 1 in the compared preparation, which indicated a marked decrease in the tetrapeptide moieties crosslinking the main glycan strands. Together with the general lower content of murein, this modification may be responsible for the enhanced sterol permeation through the cell wall.