We have sequenced the cDNA of a novel Ca(2+)-activated cysteine proteinase (calpain) from the fruit fly, Drosophila melanogaster. The predicted protein, designated as CALPB, shows high similarity to the previously identified Drosophila calpain, CALPA. The two proteins were expressed in Escherichia coli and purified to homogeneity by metal-chelate affinity chromatography either from inclusion bodies or from the bacterial cytosol. Both enzymes were Ca(2+)-dependent proteinases and attained half-maximal activation in the presence of millimolar Ca(2+). The activity and the rate of activation of CALPA, but not CALPB, could be activated by phosphatidylinositol 4,5-diphosphate, phosphatidylinositol 4-monophosphate, phosphatidylinositol, and phosphatidic acid. A truncated form of CALPA, lacking the CALPA-specific unique insertion region, has also been expressed and characterized. Although it lacked the 16-amino acid long putative membrane-anchoring segment, its activation by phospholipids was similar to that of the full-length CALPA protein. The enzymes undergo N-terminal autolysis in a Ca(2+)-dependent manner which was shown with CALPB to run parallel with enzyme activation. Moreover, fully autolyzed CALPB lacked the characteristic activation phase indicating the requirement for autolysis upon activation of this calpain form in vitro. The analysis of the mechanism of activation in Drosophila calpains seems to corroborate the autolysis model of calpain activation.