Little is known about the aetiology of transsexualism and both environmental and biological factors may contribute. Anatomical and MRI studies reveal that sexually dimorphic brain structures in male-to-female (MtF) transsexuals are more similar to females than males. There is a likely genetic component and sex steroidogenesis genes are good candidates. Androgen receptor (AR), aromatase (CYP19) and oestrogen receptor (ER) have been the focus of studies by we and others, with variable results. To further investigate the genetic basis of transsexualism by examining additional genes involved in sex steroidogenesis in a larger cohort. A genetic association study was conducted with 380 MtF transsexuals and 344 Caucasian male control subjects. Eight genes were analysed, seven of which have functional repeat length gene polymorphisms; androgen receptor (AR), aromatase (CYP19), oestrogen receptor β (ERβ), oestrogen receptor α (ERα), Cyp11A1, progesterone receptor (PGR) and 5-alpha reductase (5αR). Cyp17 is a T/C SNP. A χ2 test was used to analyse the number of short and long alleles in each of these genes. Logistic regression was used to compute the ORs and 95% CIs for the genotypes in all genes. Gene-gene interactions were also analysed by binary logistic regression. Significant associations were identified between transsexualism and variants in 5αR, with transsexual individuals being more likely to possess the TA(0)/TA(0) genotype than the male control subjects (P≤0.001). Associations were also identified with ERα, with transsexual individuals being more likely to have the short allele (P≤0.03). There was a higher incidence of the Cyp17 A2A2 genotype in transsexuals than the male control cohort (P≤0.04). These findings suggest a significant role for 5αR in transsexual patients. 5αR converts testosterone (T) to its more potent form dihydrotestosterone (DHT), which then binds to the AR to produce an active hormone-receptor complex. Transsexual patients with TA(0) have a shorter version of the gene, potentially leading to a lower rate of conversion of T to DHT. Minor contributions from ERα and Cyp17 are also indicated. We speculate that the consequence of the functional variants overrepresented in the MtF population may result in reduced androgen signalling in the MtF brain.