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SUMMARY: The effects of various factors on the release of penicillinase from apparently undamaged cells of a growing culture of Bacillus subtilis were investigated. The enzyme was not eluted from the cells by treating them with high concentrations of salt. Its liberation did not take place at all at 0°, and was nearly completely inhibited at pH values below 6.0, whereas chloramphenicol, at concentrations sufficient to cause complete cessation of growth, caused only partial inhibition of enzyme release. The penicillinase-releasing action of extracts containing heat-labile ‘autolytic’ factors from older cells of the same organism could not be dissociated from their damaging effect on the cell, as indicated by concomitant release of the normally intracellular α-glucosidase. It is concluded that normal penicillinase liberation is controlled by enzymic reactions, as yet unidentified, involving detachment of the enzyme from structures superficially located in the cell envelope.