We hypothesized that abnormal fetal lung growth in experimental CDH after maternal nitrofen exposure alters lung structure due to impaired VEGF signaling, which can be reversed with VEGF or NO treatment. Timed-pregnant Sprague Dawley rats were treated with nitrofen on E9, and fetal lungs were harvested for explant culture on E15. Explants were maintained in 3% O2 for 3 days, and were treated with NO gas or rhVEGF protein for 3 days. To determine the effects of VEGF inhibition on lung structure, normal fetal lung explants were treated with SU5416, a VEGF receptor inhibitor, with or without exogenous NO or VEGF. We found that nitrofen treatment impaired lung structure, as evidenced by decreased branching at day 0, but lung structure was not different from controls after 3 days in culture. Nitrofen reduced lung VEGF but not eNOS protein expression. Treatment with NO enhanced lung growth in control and nitrofen-exposed lungs, however, the response to NO in the nitrofen-treated lungs was reduced when compared to controls. VEGF treatment did not cause a further increase in lung complexity after nitrofen exposure. SU5416 treatment altered lung structure, which improved with NO but not VEGF treatment. Both nitrofen and SU5416 treatment increased apoptosis in the mesenchyme of fetal lung explants. We conclude that nitrofen exposure increased apoptosis, decreased lung growth and reduced VEGF expression, and that exogenous NO but not VEGF treatment enhances lung growth. Disruption of lung architecture after VEGF receptor blockade was similar to nitrofen-induced changes, but was more responsive to NO.