Objectives: Family represents the closest social environment that immediately affects human ontogeny from an early prenatal period. This study aimed to assess sibship influences on the second-to-fourth digit length ratio (2D:4D ratio). Methods: The source sample represented 329 children aged 6.5 to 15.8 years (Czech Republic), including a subsample of 75 sibling pairs. A combination of (a) betweenfamily design (cross-sectional sample) and (b) within-family design (pairs of siblings) was used to study the effect of family variables and interbirth interval (IBI) on the 2D:4D ratio. Results: Birth order, number of siblings (except for younger sisters), and sex ratio in siblings were significantly related to the 2D:4D ratio on the right, left, or both hands.At the same time, the relationships were opposite in males and females for birth order, number of older brothers and number of children in the family--increasing values in these variables increased digit ratio in males but decreased digit ratio in females. Mean difference in 2D:4D ratio (DIFF) within pairs (older minus younger sibling) differed from zero only in the "older sister--younger brother" group, where DIFF did not depend on IBI. On the contrary, in remaining pair types the DIFF tended to change with IBI, the strongest in the "older brother--younger sister" pairs. Conclusions: Family variables, especially number of older brothers, should be considered as important confounding factors in 2D:4D ratio studies. However, the effect of these variables might be modified by IBI.