As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside‐induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside‐induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside‐induced readthrough in the order C > U > A ≥ G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33–base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low‐level aminoglycoside‐induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed. Ann Neurol 2000;48:164–169