Probably the most robust cognitive sex difference is that, on average, males outperform females in tests of mental rotation ability (Voyer, Voyer, & Bryden, 1995). This sex difference is evident across many different cultures (Silverman, Choi, & Peters, 2007), and it appears early in infancy, at 3 to 5 months of age (Moore & Johnson, 2008; Quinn & Liben, 2008). Studies have suggested that elevated levels of fluctuating testosterone are associated with better spatial abilities among females, whereas in males, lower fluctuating testosterone levels relate to better spatial abilities (Gouchie & Kimura, 1991; Moffat & Hampson, 1996). In persons with congenital adrenal hyperplasia, which usually affects the production of sex steroids, the organizational effects of elevated prenatal testosterone may account for improved spatial test performance in females and impaired spatial test performance in males (Puts, McDaniel, Jordan, & Breedlove, 2008). Moreover, a positive relationship between mental rotation test (MRT) performance and prenatal testosterone has been reported in healthy girls (Grimshaw, Sitarenios, & Finegan, 1995). Animal studies have shown that female fetuses located adjacent to male fetuses in the uterus can be masculinized through prenatal exposure to testosterone (Ryan & Vandenbergh, 2002). In humans, prenatal masculinization can be studied by comparing female twins from opposite-sex twin pairs with female twins from same-sex pairs. Sisters of twin brothers have been reported to be masculinized in physiological traits, including maternal fitness (Lummaa, Pettay, & Russell, 2007) and second-to-fourth-finger ratio (van Anders, Vernon, & Wilbur, 2006), but other studies have not demonstrated the same results (Medland, Loehlin, & Martin, 2008; Medland, Loehlin, Willemsen, et al., 2008). One commonly cited study concerns mental rotation ability in female twins from opposite- and same-sex pairs (Cole-Harding, Morstad, & Wilson, 1988); however, this study was published only as a meeting abstract. In the study reported here, we assessed masculinization effects on MRT performance among female twins with male co-twins by comparing their performance with that of female twins with female co-twins. We also investigated whether or not there are differences in MRT performance between males with female co-twins and males with male co-twins.