Calpains, Ca(2+)-activated cysteine proteases, have been implicated in the progression of multiple disease states. We recently identified calpain 10 as a mitochondrial calpain that is involved in Ca(2+)-induced mitochondrial dysfunction. The goals of this study were to characterize the expression and activity of renal mitochondrial calpain 10 in rabbit, mouse, and rat. Using shRNA technology and immunoblot analysis three previously postulated splice variants of calpain 10 were identified (50, 56, and 75kDa). SLLVY-AMC zymography and immunoblot analysis was used to directly link calpeptin-sensitive calpain activity to calpain 10 splice variants. Rabbit, mouse, and rat kidney mitochondria contained 75kDa (calpain 10a), 56kDa (calpain 10c or 10d), and 50kDa (calpain 10e) splice variants. Interestingly, zymography yielded distinct bands of calpain activity containing multiple calpain 10 splice variants in all species. These results provide evidence that several previously postulated splice variants of calpain 10 are localized to the mitochondria in kidneys of rabbits, rats, and mice.