In rat lung, the definitive alveoli are established during development by the outgrowth of secondary septa from the primary septa present in newborn; however, the mechanism of alveolar formation has not yet been fully clarified. In this study, we characterize the septal interstitial cells in developing alveoli. During the perinatal period, alpha-SMA-containing slender cells were found in the primitive alveolar septa. Alpha-SMA-containing cells were detected at the tips of the septa until postnatal day 21, when the alveolar formation was almost completed, but disappeared in adult. Immunoelectron microscopy demonstrated that alpha-SMA is localized mainly in the cellular protrusions, which are connected with the elastic fibers around the interstitial cells. Developmentally regulated brain protein (drebrin) is also located in the cell extensions containing alpha-SMA in immature alveolar interstitial cells. In adult lung, alpha-SMA-positive cells are located only at the alveolar ducts but are not found in the secondary septa. Desmin is expressed only in alpha-SMA-containing cells at the alveolar ducts but not in those at the tip of alveolar septa. These results suggest that a part of the septal interstitial cells are temporarily alpha-SMA- and drebrin-positive during maturation. Alpha-SMA- and drebrin-containing septal interstitial cells (termed septal myofibroblast-like cells) may play an important role in alveolar formation.