BACKGROUND The calmodulin-independent pathway is thought to involve the activation of calcineurin by calpain. However, the effect of endogenous calpain on calcineurin in human heart is not well known. METHODS Proteolysis and activation of recombinant calcineurin by purified calpain isozymes I and II as well as endogenous calcineurin by calpains in the human heart were investigated by Western blot. Activation of calpain and calcineurin in the human heart was examined using zymography and a calcineurin activity assay. RESULTS Calpains I and II caused limited proteolysis of full-length calcineurin in a Ca(2+)-dependent manner, and the degradation fragment(s) were constitutively active in the absence of calmodulin. Calpain I and calcineurin were expressed in ventricular myocardium from patients with heart failure, with no difference in expression levels in the left and right heart chambers. Human heart calpains I and II degraded the specific substrate casein in gels which were incubated in medium containing Ca2+, but not in Ca(2+)-free medium. Calpain inhibitor-sensitive calcineurin activity was stimulated in the human ventricular myocardium in the presence of concentrations of Ca2+ that were found to activate calpain I. CONCLUSIONS Proteolysis of calcineurin A by endogenous calpain I leads to the formation of constitutively active calcineurin in the human heart, which may contribute to the pathogenesis of myocardial disease.