Disruption of pulmonary development caused by premature birth before the achievement of functional pulmonary maturation culminates in respiratory distress syndrome, primarily due to surfactant deficiency. Furthermore, the severity of this syndrome intensifies, particularly in the case of extremely premature neonates. This investigation aimed to evaluate the presence of postnatal pulmonary functional maturation in premature neonates. In pursuit of this objective, we conducted immunohistochemical assays for surfactant and Napsin A within the pulmonary tissue of 67 preterm neonates, with gestational ages ranging from 23 to 35 weeks, whose lifespans varied between one day and 149 days. The two immunohistochemical markers were evaluated within the pulmonary distal epithelium, and their expression was interpreted in relation to various pre- and postnatal factors. The examination was performed on tissue microarrays, sectioned at 5 micrometers, and the assessment of the immunohistochemical markers was interpreted from photographs captured under the optical microscope. Our investigation revealed that all neonates, regardless of their gestational age or lifespan, demonstrated the presence of surfactant within the pulmonary tissue. The intensity of Napsin A expression exhibited a positive correlation with gestational age, duration of oxygen therapy/mechanical ventilation, administration of antenatal corticosteroids, and maternal infections during pregnancy. In summary, our research demonstrated that mechanical ventilation, through the dynamic process of alveolar distension, promotes surfactant production within the distal lung epithelium. Antenatal treatment with corticosteroids and maternal antenatal infections enhances pulmonary function, facilitating surfactant production.