The aim of our study was to describe the effect of prenatal testosterone exposure on 2D:4D in both sexes, and to determine whether this effect is mediated via the androgen receptor. In addition, the sex differences in lengths of 2D, 4D, and 2D:4D ratio were analyzed. BACKGROUND: Clinical studies suggest a negative correlation between prenatal testosterone exposure and ratio of the lengths of the second and fourth digits (2D:4D). However, less is known about the underlying molecular mechanisms. METHODS: Pregnant rats were treated with olive oil, testosterone, fl utamide or testosterone with fl utamide daily from the fourteenth day of pregnancy until delivery. The fi nger lengths of adult offspring were measured using both, digital scanning of the paws and μCT analysis of the phalanges. RESULTS: None of the aforementioned methods revealed any effect of testosterone on 2D:4D. μCT measurements showed that prenatal hyperandrogenism in both sexes leads to shorter 2D compared to controls. Moreover, the testosterone treatment in males resulted in the shortening of 4D when compared to controls. CONCLUSION: Prenata l hyperandrogenism leads to shorter lengths of 2D and 4D; however, it does not affect 2D:4D ratio. Whether other steroid hormones and/or testosterone metabolites affect the 2D:4D ratio requires further investigation (Tab.