Cystic fibrosis (CF) is an autosomal recessive disorder caused by many types of genetic defects, including premature stop codons. Gentamicin can suppress stop mutations in CF transmembrane conductance regulator (CFTR) in vitro and in vivo, leading to improvements in CFTR-dependent ion transport and protein localization to the apical surface of respiratory epithelial cells. The primary objective of this study was to test whether nasally administered gentamicin or tobramycin could suppress premature stop mutations in CFTR, resulting in full-length, functional protein. A secondary objective was to obtain data to aid in the design of multicenter trials using the nasal potential difference as a study endpoint. A multicenter study was conducted in two cohorts of patients with CF, those heterozygous for stop mutations in the CFTR gene and those without nonsense mutations, to investigate the effects of both gentamicin and tobramycin administered over a 28-d period on sequential nasal potential difference and airway cell immunofluorescence endpoints. Eleven patients with CF with stop mutations were enrolled in a randomized, double-blinded, crossover fashion to receive each drug, while 18 subjects with CF without stop mutations were randomized 1:1 in a parallel fashion to receive one drug. After demonstration of drug delivery, neither aminoglycoside produced detectable changes in nasal ion transport or CFTR localization in brushed cells from either study group. These results with first-generation suppressive agents suggest the need for improved drug delivery methods and/or more potent suppressors of nonsense mutations to confer CFTR correction in subjects with CF heterozygous for nonsense mutations. The study provides valuable information on parameters of the nasal potential difference measurements for use in future multicenter clinical trials.