The extracellular release of mycobacillin from Bacillus subtilis first occurred in the medium at the onset of stationary phase and continued at a high rate even after 6 days. Mycobacillin synthetase activity appeared earlier than late-exponential phase in the cytosol of producer cells and was not sedimentable even at 105,000g. The activity then quickly reached the maximum late in the stationary phase. With further increase in the age of the culture, the activity gradually disappeared from the cytosol, to reappear concomitantly in the membrane in an insoluble particulate form, even in absence of protein synthesis. The membrane-bound synthetase activity was sedimentable at 10,000g and was fairly active even after 5 days.