We compared the effectiveness of antenatal betamethasone for the prevention of neonatal morbidity and mortality in preterm twin and singleton gestations. We conducted a case-control study of women with twin versus singleton gestations who received betamethasone for risk of prematurity in a university-affiliated, community-based, tertiary care center between 1997 and 2005. Cases were identified from clinical care and pharmacy databases, then matched for neonatal gender and gestational age (GA) at delivery. Sixty cases and 60 controls of deliveries occurring between 24 and 34 weeks' gestation were identified. The mean GA was 30.4 +/- 2.7 weeks. There were no differences between the groups in maternal demographics (with the exception of maternal age), birth weight, head circumference, Apgar scores, need for mechanical ventilation, days on ventilator, intraventricular hemorrhage grade 3 or 4, necrotizing enterocolitis suspected sepsis, total days in neonatal intensive care unit, or neonatal deaths. No differences in major morbidities or mortality were found in singletons versus twins. Concerns that the added maternal plasma volume in multiple gestations could lessen the neonatal benefits of antenatal betamethasone were not substantiated. This study may be affected by beta-error due to small sample size and sampling bias as a result of a retrospective study.