AbstractFoetal sex hormones can have powerful and far-reaching effects on later phenotype. However, obtaining accurate measurements is difficult for ethical reasons, and researchers often employ proxy variables to examine their effects. The relative length of the second and fourth fingers (digit ratio or 2D:4D) is frequently used for this purpose, as it is hypothesized to index variance in prenatal androgen and oestrogen exposure. Most studies employing this method examine digit ratio for the right hand (R2D:4D) and/or left hand (L2D:4D), though the mean value (M2D:4D) (i.e., the average of R2D:4D and L2D:4D) and directional asymmetry (D_[R–L]) (i.e., R2D:4D minus L2D:4D) are also commonly used. As no published studies have examined M2D:4D or D_[R-L] in relation to testosterone measured from amniotic fluid, we conducted a secondary analysis of data published by Ventura et al. The sample comprises 106 mothers from Portugal who underwent amniocentesis during the second trimester and their neonates. Newborn M2D:4D was negatively correlated with amniotic testosterone in females (P<0.05) but not in males; no significant association was observed between amniotic testosterone and D_[R–L] in either sex. In addition, we examined testosterone measured from maternal circulation during the second trimester, and found that it was not a significant predictor of M2D:4D or D_[R–L] in male or female infants. Further research should aim to measure the ratio of testosterone to oestradiol present in amniotic fluid and maternal plasma, to examine whether either is a predictor of digit ratio variables at different stages of postnatal development.