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BACKGROUND: Soft tissue measurements from the hand reveal lower second to fourth finger digit ratios (2D:4D) in males compared with females. The relatively longer 4th finger observed in males may be related to increased fetal exposure to androgens influencing the expression of Hox genes. METHODS: We have measured 2D:4D ratios in 69 healthy females [median age 9.3 (range 1.9-17) years], 77 control males [median age 13.86 (2.1-20.3) years] and in 66 females with classical virilizing congenital adrenal hyperplasia (CAH) (median age 8.5 (1.1-16.2) years] who are known to be exposed to high concentrations of androgens in utero. Measurements were determined from X-rays of the left hand using vernier callipers. Intra-observer variability in measurement technique was 0.01%. RESULTS: Control males had a significantly lower mean (SD) 2D:4D ratio [0.918 (0.029)] compared with female patients [0.927 (0.029), ANOVA P = 0.02]. No difference in 2D:4D ratio was observed between CAH females [0.925 (0.021)] and control females [0.927 (0.029)]. In contrast, 2D:4D ratio in males were significantly lower compared with CAH females (P = 0.03). CONCLUSIONS: 2D:4D ratios determined directly from radiographs of the left hand confirm significant differences between males and females. However, female patients with virilizing CAH do not have a male digit ratio pattern suggesting that in the left hand digit ratio development is not influenced by in-utero exposure to androgens. [ABSTRACT FROM AUTHOR]