Calpain is a Ca²⁺‐dependent cysteine proteinase that has neutral pH optima. There are two classes of calpains that differ in their optimal calcium ion concentration for enzymatic activity. Calpain I requires a low concentration of Ca²⁺ for activation, and calpain II requires a much higher Ca²⁺ concentration. This report describes the immunohistochemical and biochemical demonstration of calpain II in calcifying cartilage in rats and also the degradation of the cartilage proteoglycan subunit by calpain II. Immunoperoxidase (peroxidase‐antiperoxidase) staining of the frozen sections of the knee joint from 3‐day‐old and 6‐day‐old Wistar rats, using polyclonal antibodies against the respective heavy subunits of calpains I and II, showed positive staining only with the anti‐calpain II antibody in the hypertrophic chondrocytes and surrounding cartilaginous matrix of the growth cartilage. Diethylaminoethyl‐cellulose chromatography of the cartilaginous extract from 3‐day‐old rats showed a peak of caseinolytic activity attributable to calpain as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anti‐calpain II antibody of the calpain peak demonstrated identity with the heavy subunit of calpain II (80 kDa). Proteoglycan‐degrading activity of calpain was assessed using porcine kidney calpain II and the porcine articular cartilage proteoglycan subunit. After incubation in the presence of Ca²⁺, degradation of proteoglycan was demonstrated by the change of the elution position on Sepharose‐2B chromatography. It is possible that calpain functions as one of the proteoglycan‐degrading proteolytic enzymes of growth cartilage. Intracellular localization of calpain in hypertrophic chondrocytes also suggests a role in the hypertrophic process of the chondrocyte in growth cartilage.