Cystic fibrosis (CF) is characterized by the presence of a viscoelastic mucus layer in the upper airways and bronchi. The underlying problem is a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein. Clinical studies of gene transfer for CF are ongoing. For gene delivery to the airways of CF patients to be effective, the mucus covering the target cells must be overcome. We therefore examined the extent to which CF sputum presents a physical barrier to the transport of nanospheres of a size comparable to that of lipoplexes and other transfection systems currently being clinically evaluated for CF gene therapy. We observed that an extremely low percentage of nanospheres (< 0.3%) moved through a 220-microm-thick CF sputum layer after 150 min. The largest nanospheres studied (560 nm) were almost completely blocked by the sputum, whereas the smaller nanospheres (124 nm) were retarded only by a factor of 1.3 as compared with buffer. Surprisingly, the nanospheres diffused significantly more easily through the more viscoelastic sputum samples. We hypothesize that the structure of the network in sputum becomes more macroporous when the sputum becomes more viscoelastic. Sputum from a patient with chronic obstructive pulmonary disease retarded the transport of nanospheres to the same extent as did CF sputum. When directly mixed with CF sputum, recombinant human deoxyribonuclease I moderately facilitated the transport of nanospheres through CF sputum.