Inhibition of the mitochondrial pathway of apoptosis has been implicated as a mechanism contributing to carcinogenesis. Chronic inflammation, which is accompanied by activation of inducible nitric oxide synthase and generation of nitric oxide (NO), is associated with cancer development in a variety of gastrointestinal diseases, including cholangiocarcinoma. Therefore, we examined the effects of NO on the mitochondrial pathway of apoptosis in human cholangiocarcinoma cell lines. Transfection with inducible NO synthase inhibited etoposide-induced apoptosis. S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), a pharmacological NO donor, did not prevent mitochondrial cytochrome c release as assessed by immunoblot analysis or cellular localization of cytochrome c-green fluorescent protein. In contrast, SNAP did prevent activation of caspase 9 in etoposide-treated cells. Furthermore, SNAP also blocked caspase 9 activation in a cell-free system and reversibly inhibited catalytic activity of human recombinant caspase 9. As assessed by the Saveille reaction, immunoprecipitated procaspase 9 from SNAP-treated cells released 6-fold more NO than untreated cells, confirming that cellular procaspase 9 is susceptible to nitrosylation. In conclusion, NO inhibits apoptosis downstream of cytochrome c release by directly blocking caspase 9 activation.