The efficiency and localization of gene transfer for gene therapy in the lung will depend on vector selection and technique used to deliver the vector. We hypothesized that surfactant might facilitate gene transfer because of its biophysical properties that enhance spreading of saline suspensions in the airways. We used a replication defective adenovirus directing the expression of the luciferase reporter gene at different virus doses and surfactant concentrations and instilled 4 ml suspensions into the lungs of adult rabbits. Transgene expression measured as luciferase-dependent light units in tissue homogenates 3 days after instillation had similar lobar distributions for surfactant-viral suspensions and for phosphate-buffered saline viral suspensions. However, total light units were four- to six-fold increased for viral doses of < 10(10) plaque forming units in 10 mg/ml or 25 mg/ml surfactant suspensions. The percentage of total transgene expression in the tracheacarina-bronchus decreased from over 50% to about 10% for virus instilled with surfactant concentrations of 2 mg/ml or greater, indicating preferential expression in airway epithelial cells located more distally in the lung. Surfactant did not enhance persistence of transgene expression 7 or 14 days after instillation. Surfactant facilitates adenovirally mediated gene transfer in the peripheral lung and enhances transgene expression.