Cell-matrix interactions have been shown to regulate the development of the lung, particularly airway branching and alveolarization. Fibronectin is the major constituent of pulmonary extracellular matrix and exists in multiple isoforms arising from alternative RNA splicing. EDA and EDB are the two major alternatively spliced segments, the expression of which is regulated in a spatiotemporal and oncodevelopmental manner. In this study, we investigated immunohistochemically the distribution of the EDA- and EDB-containing fibronectin isoforms (referred to as EDA+ fibronectin and EDB+ fibronectin, respectively) in normal and hypoplastic human lungs at different gestational ages to explore the role of these fibronectin isoforms in alveolarization. EDA+ fibronectin was expressed around the distal airspaces throughout the development of both normal and hypoplastic lungs. In contrast, the expression of EDB+ fibronectin was restricted to the lung with morphologically immature acinar complex, typically observed in normally developing lungs of < 30 gestational weeks or in hypoplastic lungs. To further confirm the restricted expression of EDB+ fibronectin in immature acinar complex, we examined the correlation of EDB+ fibronectin expression with that of the surfactant protein SP-A, a biochemical marker for the differentiated type II pneumocytes. A clear inverse relationship between the immunoreactivities for EDB+ fibronectin and SP-A was observed in both control and hypoplastic lungs. Given the proposed importance of fibronectins in the differentiation of alveolar epithelial cells, our results suggest that the EDB segment plays a regulatory role in the differentiation of immature acinar epithelial cells into type II pneumocytes. The EDB segment may also serve as a new histochemical marker for the functional maturity of fetal lung tissues.