Congenital diaphragmatic hernia (CDH) is currently the most life-threatening congenital anomaly which major finding is lung hypoplasia. Lung hypoplasia pathophysiology involves early developmental molecular insult in branching morphogenesis and a late mechanical insult by abdominal herniation in maturation and differentiation processes. Since early determinants of lung hypoplasia might appear as promising targets for pre-natal therapy, proteomics analysis of normal and nitrofen-induced hypoplastic lungs was carried out at 17.5 days post-conception. The major differentially expressed protein was identified by mass spectrometry as myosin light chain 1a (MLC1a). Embryonic essential MLC1a and regulatory myosin light chain 2 (MLC2) were characterized throughout normal and abnormal lung development by immunohistochemistry and western blot. Disruption of MLC1a expression was assessed in normal lung explant cultures by antisense oligodeoxynucleotides. Since early stages of normal lung development, MLC1a was expressed in vascular smooth muscle (VSM) cells of pulmonary artery and MLC2 was present in parabronchial smooth muscle and VSM cells of pulmonary vessels. Additionally, early smooth muscle differentiation delay was observed by immunohistochemistry of alpha-smooth muscle actin and transforming growth factor beta 1. Disruption of MLC1a expression during normal pulmonary development led to significant growth and branching impairment, suggesting a role in branching morphogenesis. Both MLC1a and MLC2 were absent from hypoplastic fetal lungs during pseudoglandular stage of lung development, whereas their expression partially recovered by prenatal treatment with vitamin A. Thus, a deficiency in contractile proteins MLC1a and MLC2, might have a role amongst the early molecular determinants of lung hypoplasia in nitrofen-induced CDH rat model.