Aminoacyl-tRNA synthetases prevent mistranslation, or genetic code ambiguity, through specialized editing reactions. Mutations that disrupt editing in bacteria adversely affect cell growth and viability, and recent work in the mouse supports the idea that translational errors caused by an editing defect lead to a neurological disease-like phenotype. To further investigate the connection of mistranslation to cell pathology, we introduced an inducible transgene expressing an editing-deficient valyl-tRNA synthetase into mammalian cells. Introducing mistranslation precipitated a disruption of cell morphology and membrane blebbing, accompanied by activation of caspase-3, consistent with an apoptotic response. Addition of a noncanonical amino acid that is misactivated, but not cleared, by the editing-defective enzyme exacerbated these effects. A special ambiguity-detecting sensor provided direct readout of mistranslation in vivo, supporting the possibility that decreased translational fidelity could be associated with disease.