We have previously shown that carbon monoxide (CO) (250 ppm) prevented tumor necrosis factor-alpha (TNF alpha)-induced apoptosis and activated the transcription factor NF-kappa B in hepatocytes both in vivo and in vitro. These studies were conducted to further determine the mechanisms by which CO suppresses apoptotic signaling in TNF alpha (10 ng/ml) and Actinomycin D (ActD, 200 ng/ml)-treated hepatocytes. Consistent with our previous findings, CO protected against TNF alpha/ActD-induced cell death, which is in part dependent on NF-kappa B activation. This was associated with a reduction in mitochondrial damage, a decrease in cytochrome c release, and an inhibition of translocation of Bel proteins to mitochondria. In conjugation with inhibition of these mitochondrial events, CO also suppressed caspases-8 and -3 cleavage in response to TNF alpha/ActD. Inhibition of NF-kappa B activation resulted in diminished CO-induced cFLIP expression and increased caspase-8 cleavage from cells treated with TNF alpha/ActD. These data indicate that CO interferes with apoptotic signaling at a proximal step. (c) 2006 Published by Elsevier Inc.