Neonatal ethanol (EtOH) exposure is associated with central nervous system dysfunction and neurotoxicity in rats. Increases in polyamine levels have been implicated as one underlying mechanism for some of EtOH's effects on the developing brain. In this study we addressed whether the inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) could reduce behavioral deficits induced by early EtOH exposure. Male and female rat pups received ethanol (6 g/kg/day EtOH i.g.), or isocaloric maltose (control) from postnatal days (PND) 1-8. On PND 8, animals were injected with either saline or DFMO (500 mg/kg, s.c.) immediately following the final neonatal treatment. Subjects were tested for isolation-induced ultrasonic vocalizations (USV) on PND 16; spontaneous activity in an open field apparatus on PND 20 and 21; and balance on PND 31. Animals exposed to EtOH neonatally displayed an increased latency to the first USV and reduced frequencies of USV, hyperactivity and preference for the center of the open field and poorer balance relative to controls. DFMO minimized these deficits in latency to the first USV and balance. These data provide further support that polyamines play a role in some of the functional deficits associated with EtOH exposure during early development and that reducing polyamine activity can improve outcome.