We have previously shown that carbon monoxide (CO) (250 ppm) prevented tumor necrosis factor-alpha (TNFalpha)-induced apoptosis and activated the transcription factor NF-kappaB in hepatocytes both in vivo and in vitro. These studies were conducted to further determine the mechanisms by which CO suppresses apoptotic signaling in TNFalpha (10 ng/ml) and Actinomycin D (ActD, 200 ng/ml)-treated hepatocytes. Consistent with our previous findings, CO protected against TNFalpha/ActD-induced cell death, which is in part dependent on NF-kappaB activation. This was associated with a reduction in mitochondrial damage, a decrease in cytochrome c release, and an inhibition of translocation of Bcl proteins to mitochondria. In conjugation with inhibition of these mitochondrial events, CO also suppressed caspases-8 and -3 cleavage in response to TNFalpha/ActD. Inhibition of NF-kappaB activation resulted in diminished CO-induced cFLIP expression and increased caspase-8 cleavage from cells treated with TNFalpha/ActD. These data indicate that CO interferes with apoptotic signaling at a proximal step.