Lung disease is the major cause of death in individuals suffering from cystic fibrosis (CF), with abnormal lung-lining fluids occurring already in early infancy. However the precise aetiology of CF lung disease is still poorly understood. We investigated the structural components of the airway surface-lining layer in targeted Cftr^tm1HGU/Cftr^tm1HGU mutant mice and non-CF controls. Five lungs per animal group were fixed by intravascular triple perfusion. The ultrastructure of the surface-lining layer of large and small intrapulmonary conducting airways was systematically investigated according to a standard protocol in transmission and scanning electron micrographs. In both animal groups, the surface-lining layer consisted of an aqueous phase and an osmiophilic film of variable thickness at the air-fluid interface. The aqueous phase did usually extend less than 1µm beyond the uppermost tips of the epithelial cells in both animal groups. The aqueous phase of the small airways was slightly more electron dense in Cftr^tm1HGU/Cftr^tm1HGU than in non-CF mice. Neither the ultrastructure of the surfactant film at the air-fluid interface nor the forms assumed by the osmiophilic structures associated with surfactant turnover in the aqueous layer differed significantly in Cftr^tm1HGU/Cftr^tm1HGU and non-CF mice. Hence, there were no signs of any ultrastructural abnormalities in the surface-lining layer of young adult Cftr^tm1HGU/Cftr^tm1HGU mice before infection with CF-related pathogens.