beta-Sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683 results in the formation of androstene derivatives and is increased in the presence of glycine. As the sterol transformation is carried out inside the cell, higher product accumulation could indicate faster diffusion of highly hydrophobic substrate through the cell wall permeability barrier. Cell wall preparations were obtained to analyse the effect of glycine on peptidoglycan components. Peptidoglycan is known to be the target for glycine action. In glycine-treated preparations, the molar ratio of diaminopimelic acid:muramic acid, the marker compounds of tetrapeptides and glycan strands respectively, was about 60% lower than in the control. This indicates a possible reduction in cross-linking between peptide units and the destruction of peptidoglycan. Unexpectedly, glycine also caused changes in the relative proportion of mycolic acids to other lipids occurring in the strain used for this study. The enhancement of beta-sitosterol side chain degradation is likely to result from disturbing the integrity of the cell wall components responsible for the permeability barrier in mycobacteria.