Despite the widespread use of antenatal glucocorticosteroids (GCs), the possibility of adverse effects on the immune response in preterm neonates remains a major concern. GCs stimulate lymphocyte apoptosis, resulting in lymphopenia and functional disorders, which have been associated with sepsis-related death in critically ill neonates. We sought to assess the effect of antenatal betamethasone (BM) on lymphocyte apoptosis in preterm neonates. Fifty preterm neonates exposed to antenatal BM and 50 controls were studied prospectively. Lymphocyte apoptosis was assessed using the annexin-V/propidium iodide (PI) assay, analysis of cell cycle after staining with PI, and intracellular caspase-3 activity. The two groups did not differ significantly as regards absolute lymphocyte counts and the percentage of lymphocytes being annexin-V (+)/PI (-) (early apoptotic) or lymphocytes in the subG1 peak after staining with PI and those with intracellular caspase-3 activation. The lymphocyte number and apoptosis were not associated with the time elapsed between antenatal BM administration and delivery. A single course of antenatal BM does not influence apoptosis of neonatal lymphocytes. This is of significant importance with respect to the preservation of lymphocyte-associated immune response in preterm neonates.