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.