This study aimed to examine the relationship between the ratio of the length of the second and fourth digits (2D:4D) and locomotor muscle strength. Furthermore, two putative mechanisms that might explain any relationship of 2D:4D with muscle strength, specifically serum total and free testosterone, and androgen receptor genotype CAG repeat number (AR CAGn) were investigated. Seventy-seven healthy young Caucasian men completed a thorough assessment of isometric and isokinetic knee extensor strength, with unilateral measurements averaged across both legs and repeated on two occasions. The lengths of the second and fourth fingers of each hand were measured to calculate 2D:4D ratio. Serum total testosterone (TT) and serum hormone binding globulin (SHBG) were measured by ELISA and used to calculate free testosterone (FT). AR CAGn was determined by PCR and microchip electrophoresis. There was no association between mean, left or right hand 2D:4D and isometric or isokinetic knee extensor strength (all, R < 0.12, P > 0.32). TT and FT were unrelated to mean, left or right hand 2D:4D ratio (all, R < 0.12, P > 0.34). Finally AR CAGn was not associated with mean, right or left hand 2D:4D ratio (all, R < 0.20, P > 0.10). This study found no evidence of 2D:4D being related to locomotor muscle strength, TT, FT, or AR CAGn. The reported association of 2D:4D with sports performance does not seem to be explained by an influence on locomotor muscle strength, and could be due to an effect on motor or cognitive skills. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.