Calpain B is one of the two calpain homologues in Drosophila melanogaster that are proteolytically active. We studied its activation by Ca2+ both in vitro and in vivo, in Schneider (S2) cells. Activation involves the autolytic cleavage, at two major sites, of the N-terminal segment, the length of which was earlier underestimated. Site-directed mutagenesis at the autolytic sites did not prevent autolysis, but only shifted its sites. Calpain B mRNA was detectable in all developmental stages of the fly. In situ hybridization and immunostaining showed expression in ovaries, embryo and larvae, with high abundance in larval salivary glands. In S2 cells, calpain B was mainly in the cytoplasm and upon a rise in Ca2+ the enzyme adhered to intracellular membranes.