Abstract We studied the effect of calcium cation on protease activity in the crude cytoplasm of Bacillus megaterium cells treated with netropsin, an antibiotic which suppresses sporulation. Only negligible proteolytic activity was found at the currently used 1 mM CaCl 2 in spite of the presence of a high concentration of Ca 2+-dependent serine protease ISP1. The Ca 2+-mediated activation of this protease in the cytoplasmic preparation was perceptible only at [Ca 2+]>5 mM and reached a maximum at [Ca 2+]>20 mM. This `delay' in activation (in terms of Ca 2+ concentration) was reduced by diluting the cytoplasmic preparation or purifying ISP1. Kinetic studies of the proteolytic reaction in the crude cytoplasm suggested that the activation was caused by Ca 2+-dependent modification of the native protease molecule. Immunological detection revealed Ca 2+-induced sequential processing of the ISP1 molecule to shorter active form(s). The suppression of this processing at lower Ca 2+ concentrations was probably caused by the presence of Ca 2+-binding compounds that chelated calcium. Their M r in the cytoplasmic preparation was lower than 3000. Possible involvement of these compounds in regulation of ISP1 activity in vivo is discussed.