We have previously demonstrated abrogation of bile salt-induced apoptosis by cathepsin B inhibitors. However, caspases have been strongly implicated in apoptosis, and the mechanistic interface between caspase and cathepsin B activation is unclear. Thus our aims were to determine the mechanistic relationship between caspases and cathepsin B in bile salt-induced apoptosis in a rat hepatoma cell line. Expression of cystatin A was used to inhibit cathepsin B, whereas Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used to inhibit caspases. Cystatin A expression prevented cathepsin B activation and apoptosis during treatment with glycochenodeoxycholate (GCDC), a toxic bile salt. CaspaseN-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) hydrolytic activity increased in both wild-type and cystatin A-transfected cells treated with GCDC, demonstrating caspase activation despite inhibition of cathepsin B. In contrast, Z-VAD-FMK blocked both DEVD-AMC hydrolytic activity and cathepsin B activity during GCDC treatment. Our data demonstrate that1) bile salt-induced apoptosis can be inhibited by the cystatin A transgene and2) caspase and cathepsin B activation are linked mechanistically with cathepsin B downstream of caspases.