Significance Calpains are a family of intracellular proteases that have in common their dependence on Ca 2+ and their ability to make a small number of specific cuts in their natural protein substrates. Although many calpain-generated protein fragments have been identified, understanding their functions is just beginning. This study shows that natural C-terminal fragments of a variety of proteins, including transmembrane ion channels, transcriptional regulators, apoptosis controllers, kinases, and phosphatases, are short-lived substrates of the N-end rule pathway, a ubiquitin-dependent processive proteolytic system that recognizes destabilizing N-terminal residues of protein substrates. This advance illuminates functional aspects of specific calpain fragments and suggests a specific molecular basis for many of the previously identified, mechanistically unclear roles of the N-end rule pathway.