Studies of singletons suggest that right-handed individuals may have higher levels of testosterone than do left-handed individuals. Prenatal testosterone levels are hypothesised to be especially related to handedness formation. In humans, female members from opposite-sex twin pairs may experience elevated level of prenatal exposure to testosterone in their intrauterine environment shared with a male. We tested for differences in rates of left-handedness/right-handedness in female twins from same-sex and opposite-sex twin pairs. Our sample consisted of 4736 subjects, about 70% of all Finnish twins born in 1983–1987, with information on measured pregnancy and birth related factors. Circulating testosterone and estradiol levels at age 14 were available on 771 and 744 of these twins, respectively. We found significantly (p = .006) lower prevalence of left-handedness in females from opposite-sex pairs (5.3%) compared to females from same-sex pairs (8.6%). The circulating levels of neither testosterone nor estradiol related to handedness in either females or males. Nor were there differences in circulating testosterone or estradiol levels between females from opposite-sex and same-sex twin pairs. Birth and pregnancy related factors for which we had information were unrelated to handedness. Our results are difficult to fully explain by postnatal factors, but they offer support to theory that relates testosterone to formation of handedness, and in a population-based sample, are suggestive of effects of prenatal testosterone transfer.