Nonsense-mediated mRNA decay (NMD) is a process of mRNA surveillance that degrades transcripts harboring a premature termination codon (PTC). Mammalian NMD was mostly studied in cultured cells so far and there was no direct evidence yet that NMD could operate in the brain. We introduced, by homologous recombination in mouse, a PTC in the mu opioid receptor gene (mor). mor transcript was severely downregulated in the brain of these knock-in mice. A systemic cycloheximide treatment significantly increased the level of the mutant mRNA, suggesting NMD involvement. To further corroborate this hypothesis, we generated a second knock-in mouse line where the PTC was placed at 10 instead of 96 nucleotides from the downstream splice junction. As predicted by the "termination codon position rule" established in vitro, mor transcript brain expression was rescued to wild-type level. These knock-in mouse lines will be valuable models to better understand and manipulate NMD in vivo.