(Rac)-Calpain Inhibitor XII is a reversible and selective inhibitor of calpain I (μ-calpain, Ki=19 nM). (Rac)-Calpain Inhibitor XII has lower affinities for calpain II (m-calpain, Ki=120 nM) and cathepsin B (Ki=750 nM). (Rac)-Calpain Inhibitor XII has the potential for studying the role of calpains in diverse processes, including neutrophil chemotaxis, neuronal signaling, and cardiac response to injury.