Effective gene transfer to the airway epithelial cells of individuals with cystic fibrosis (CF) requires gene therapy vectors to effectively penetrate the mucous lining of the airways of these patients. In this study, we examined the effects of the aqueous sol fraction of sputum recovered from CF patients (CF sol) on adenovirus (Ad)-mediated gene transfer to cultured epithelial cells. Sputum collected from patients with CF was separated into aqueous sol and gel fractions by ultracentrifugation and the sol fraction from different individuals was pooled. To determine if CF sol affects Ad-mediated transfection, Fisher rat thyroid (FRT) epithelial cells or normal human bronchial epithelial (NHBE) cells were infected with an Ad encoding beta-galactosidase (Ad2/betagal-2) in the presence or absence of the pooled CF sol. Transfection efficiency was determined by measuring beta-Gal activity. CF sol significantly inhibited Ad2-mediated gene transfer in a dose-dependent manner when the vector was incubated with CF sol prior to exposure to the cells. In contrast, preincubation of the cells with the sol was without effect. The inhibition of Ad-mediated gene transfer by CF sol was not related to its low pH, was abrogated by preadsorption with an Ad2 serotype vector, and was neutralized by heat treatment, but was not affected by treatment with protease inhibitors. Analysis of CF sol fractions from seven different individuals with CF showed inhibition of Ad-mediated gene transfer in four of the seven samples tested and, further, the inhibitory effect was correlated with the presence of Ad-specific antibodies. We conclude that preexisting adenovirus-specific antibodies present in some of the patient samples were the predominant factor inhibiting Ad-mediated gene transfer.