Increased prenatal exposure to testosterone (T) in females of an opposite-sex (OS) twin pair may have an effect on the development of sex-typical cognitive and behavioral patterns. The prenatal exposure to T due to hormone transfer in OS twin females may occur in two ways, one directly via the feto–fetal transfer route within the uterus, the other indirectly through maternal–fetal transfer and based in the maternal–fetal compartment. Although some studies in singletons indeed found that women pregnant with a male fetus have higher T levels during gestation than women pregnant with a female fetus, many other studies could not find any relation between the sex of the fetus and maternal serum steroid levels. Therefore at present it is unclear whether a pregnant woman bearing a male has higher levels of T than a woman bearing a female. Up to this point, no-one has investigated this issue in twin pregnancies. We examined the relationship between maternal serum steroid levels and sex of fetus in 17 female–female, 9 male–male and 29 OS twin pregnancies. No differences were observed between the maternal serum steroid levels of women expecting single-sex and mixed-sex offspring. It is concluded that the source of prenatal T exposure in females probably comes from the fetal unit, which is the direct route of fetal hormone transfer.