Although it is clearly established that surfactant protein A (SP-A) is secreted by type II pneumocytes as a component of pulmonary surfactant, its secretion pathway as well as its subcellular localization in the human lung are uncertain. We therefore studied the intracellular and intra-alveolar localization of SP-A in eight adult human lungs by immunohistochemistry and immunoelectron microscopy. Only type II pneumocytes could be identified as SP-A positive cells within the parenchymal region. SP-A was localized mainly in small vesicles and multivesicular bodies close to the apical plasma membrane. Only few lamellar bodies were weakly labeled at their outer membranes. Stereologic analysis showed this weak signal to be due to specific labeling. In the alveolar space, lamellar body-like surfactant forms in close proximity to tubular myelin were labeled for SP-A at their periphery. The strongest SP-A labeling was found over tubular myelin figures. Labeling for SP-A was also found in close association with the surface film and unilamellar vesicles. Our results support the hypothesis that, in the human lung, SP-A is mainly secreted into the alveolar space via an alternative pathway that largely bypasses the lamellar bodies. After secretion, the outer membranes of unwinding lamellar bodies become enriched with SP-A when tubular myelin formation is initiated. SP-A may also be involved in the transition of tubular myelin into the surface film.