Objective: Brain volume of boys is larger than that of girls by w10%. Prenatal exposure to testosterone has been suggested in the masculinization of the brain. For example, in litter-bearing mammals intrauterine position increases prenatal testosterone exposure through adjacent male fetuses, resulting in masculinization of brain morphology. Design: The influence of intrauterine presence of a male co-twin on masculinization of human brain volume was studied in 9-year old twins. Methods: Magnetic resonance imaging brain scans, current testosterone, and estradiol levels were acquired from four groups of dizygotic (DZ) twins: boys from same-sex twin-pairs (SSM), boys from opposite-sex twin-pairs (OSM), girls from opposite-sex twin-pairs (OSF), and girls from same-sex twinpairs (SSF; nZ119 individuals). Data on total brain, cerebellum, gray and white matter volumes were examined. Results: Irrespective of their own sex, children with a male co-twin as compared to children with a female co-twin had larger total brain (C2.5%) and cerebellum (C5.5%) volumes. SSM, purportedly exposed to the highest prenatal testosterone levels, were found to have the largest volumes, followed by OSM, OSF and SSF children. Birth weight partly explained the effect on brain volumes. Current testosterone and estradiol levels did not account for the volumetric brain differences. However, the effects observed in children did not replicate in adult twins. Conclusions: Our study indicates that sharing the uterus with a DZ twin brother increases total brain volume in 9-year olds. The effect may be transient and limited to a critical period in childhood.