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From early childhood, gender identity and the 2nd to 4th finger length ratio (2D:4D) are discriminative characteristics between sexes. Both the human brain and 2D:4D may be influenced by prenatal testosterone levels. This calls for an examination of 2D:4D in patients with gender identity disorder (GID) to study the possible influence of prenatal testosterone on gender identity. Until now, the only study carried out on this issue suggests lower prenatal testosterone levels in right-handed male-to-female GID patients (MtF). We compared 2D:4D of 56 GID patients (39 MtF; 17 female-to-male GID patients, FtM) with data from a control sample of 176 men and 190 women. Bivariate group comparisons showed that right hand 2D:4D in MtF was significantly higher (feminized) than in male controls, but similar to female controls. The comparison of 2D:4D ratios of biological women revealed significantly higher (feminized) values for right hands of right handed FtM. Analysis of variance confirmed significant effects for sex and for gender identity on 2D:4D ratios but not for sexual orientation or for the interaction among variables. Our results indirectly point to the possibility of a weak influence of reduced prenatal testosterone as an etiological factor in the multifactorially influenced development of MtF GID. The development of FtM GID seems even more unlikely to be notably influenced by prenatal testosterone.