Cathepsin D, a lysosomal aspartic proteinase, has been shown to induce apoptosis of HeLa cells when overexpressed. To further understand regulatory mechanisms of cathepsin D-induced cell death, we examined whether lysosomal cysteine and aspartic proteinases are involved in apoptosis of PC12 cells following serum deprivation. In serum deprived culture, PC12 cells overexpressing cathepsin D died more rapidly than wild-type cells. When the active forms of cathepsins B and D were examined during the apoptotic process of wild-type cells, the amount of cathepsin B was drastically reduced 24 hr after the onset of culture, whereas that of cathepsin D considerably increased. The viability of PC12 cells overexpressing cathepsin B was significantly higher in serum-deprived culture than wild-type cells. In this situation, the amount of the cathepsin B protein did not decrease. The results suggest that there exists an apoptotic pathway regulated by lysosomal cathepsins B and D.