Male and female twins with opposite-sex co-twins were compared to twins with same-sex co-twins on three independent dimensions of masculinity-femininity, in order to examine the hypothesis that the hormones of the co-twin might have an effect on prenatal masculinization. The analysis was originally carried out for an older cohort from the Australian Twin Registry (2647 pairs, mean age 41.2), and then repeated in a younger cohort (1503 pairs, mean age 23.2). For women, the results on two of the three scales support and extend that of an earlier large study in Finland by Rose et al. (1994), who found no effect of sex of co-twin on feminine interests. One of the two scales contrasted worried and calm individuals, the other, confiding and reserved ones. The third scale, willingness to break or bend rules, showed a small effect of shared environmental influence, and it lay in the expected direction for a prenatal hormonal effect--females with a male co-twin scored higher (more like males). Most previous studies have not looked at the effect of sex of co-twin on males. The present study detected several such effects, although all were small in magnitude. The pattern was complex: sometimes the effect was in the masculine direction, sometimes in the feminine direction; sometimes there was agreement between the older and younger cohorts, sometimes not. Overall, the results suggest that no simple masculinization hypothesis--prenatal or postnatal--will adequately account for all the evidence. Age, sex, and aspect of masculinity-femininity must be taken into account.