Hematopoietic cells bearing inactivating mutations of Fanconi anemia group C (FANCC) are excessively apoptotic and demonstrate hypersensitivity not only to cross-linking agents but also to interferon gamma (IFN-gamma) and tumor necrosis factor-alpha. Seeking essential signaling pathways for this phenotype, this study quantified constitutive and induced RNA-dependent protein kinase (PKR) activation in Fanconi anemia cells of the C complementation group (FA-C). PKR was constitutively phosphorylated and exhibited an increased binding affinity for double-stranded RNA (dsRNA) in FANCC(-/-) cells. FANCC(-/-) cells were hypersensitive to both dsRNA and the combination of dsRNA and IFN-gamma in that these agents induced a higher fraction of apoptosis in FANCC(-/-) cells than in normal cells. Overexpression of wild-type PKR-sensitized FANCC(-/-) cells to apoptosis induced by IFN-gamma and dsRNA. Conversely, inhibition of PKR function by enforced expression of a dominant-negative inhibitory mutant of PKR (PKRDelta6) substantially reduced the IFN and dsRNA hypersensitivity of FANCC(-/-) cells. Two PKR target molecules, IkappaB-alpha and IRF-1, were not differentially activated in FANCC(-/-) cells, but enforced expression of a nonphosphorylatable form of eukaryotic translation initiation factor-2alpha reversed the PKR-mediated block of messenger RNA translation and partially abrogated the PKR-mediated apoptosis in FANCC(-/-) cells. Because no evidence was found of a PKR/FANCC complex in normal cells, it was concluded that an essential function of FANCC is to suppress, indirectly, the activity of PKR and that FANCC inactivation results in IFN hypersensitivity, at least in part, because this function of FANCC is abrogated.