Male and female mice are differentially sensitive to the effects of alcohol on aggressive behavior. We investigated the role of testosterone during sexual differentiation in determining sex differences in alcohol effects on aggression. On the day of birth male mice were castrated or sham-operated. Neonatal female mice were injected with 250 μg of testosterone propionate (TP) or the oil vehicle. At approximately 75 days of age the mice which had not been gonadectomized at birth were gonadectomized. Control males and androgenized female mice then received 7.5 mm Silastic capsules containing testosterone, SC. Aggressive behavior toward an intruder was assessed following administration of ethanol (0.1–3.0 g/kg) or water, PO. Neonatally sham-gonadectomized male mice had a significant increase in aggressive behavior following administration of 1.0 g/kg alcohol, with no significant suppression of aggression at 3.0 g/kg. Neonatally androgenized female mice showed neither the male-typical response to adult testosterone and alcohol, nor did they show the female-typical response. Neonatally gonadectomized males showed an alcohol dose response curve that was similar to that of androgenized females. Postnatal testosterone did not appear to completely determine the male- and female-typical responses to alcohol on aggression. The critical period for this sexually dimorphic response to alcohol and testosterone may be primarily prenatal.