The functional significance of sex differences in the size of the corpus callosum was investigated using a prenatal alcohol exposure paradigm that influences the fetal hormonal milieu. Adult male and female Long-Evans rats were selected from one of 3 prenatal treatment histories: prenatal alcohol-exposed (35% ethanol-derived calories), nutritional control (0% ethanol-derived calories) or standard control (lab chow). Subjects were assessed for open field activity at 85 days of age. At 100 days of age, midline sagittal areas of the corpus callosum and the anterior commissure were determined for these subjects. Male control subjects had significantly larger callosal areas than female controls. Prenatal alcohol exposure significantly decreased the total callosal area, and abolished this sexual dimorphism. When the callosal measurements were analyzed using Denenberg's regions, differential effects of prenatal alcohol exposure, undernutrition and sex were dissociable by subarea. There were no significant sex differences or effects of prenatal alcohol exposure in the midline sagittal area of the anterior commissure. Callosal size was negatively correlated to open field activity, suggesting a possible role in normal exploratory behavior and to the overactivity observed after prenatal alcohol exposure.