Under physiological conditions, manganese(II) exhibits catalase-like activity. However, at elevated concentrations, it induces apoptosis via a non-mitochondria-mediated mechanism (Oubrahim, H., Stadtman, E. R., and Chock, P. B. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9505-9510). In this study, we show that the Mn(II)-induced apoptosis, as monitored by caspase-3-like activity, in NIH3T3 cells was inhibited by calpain inhibitors I and II or the p38 MAP kinase inhibitor, SB202190. The control experiments showed that each of these inhibitors in the concentration ranges used exerted no effect on activated caspase-3-like activity. Furthermore, caspase-12 was cleaved in Mn(II)-treated cells, suggesting that the Mn(II)-induced apoptosis is mediated by caspase-12. This notion is confirmed by the observations that pretreatment of NIH3T3 cells with either caspase-12 antisense RNA or dsRNA corresponding to the full-length caspase-12 led to a dramatic decrease in caspase-3-like activity induced by Mn(II). The precise mechanism by which Mn(II) induced the apoptosis is not clear. Nevertheless, Mn(II), in part, exerts its effect via its ability to replace Ca(II) in the activation of m-calpain, which in turn activates caspase-12 and degrades Bcl-xL. In addition, the dsRNA(i) method serves as an effective technique for knocking out caspase-12 in NIH3T3 cells without causing apoptosis.