There is evidence that the digit ratio (2D:4D) is a negative correlate of prenatal levels of testosterone, but there is no association between 2D:4D and the circulating levels of both total and free testosterone. Sports provide a physical challenge and participants often show increased levels of free testosterone immediately preceding and during competition. We tested this hypothesis of a link between 2D:4D and testosterone under challenge in 79 professional rugby players using the following procedures; (i) 25 players were physically challenged using a repeated sprint agility test, and saliva samples were assayed for testosterone immediately preceding the repeated sprint agility test (time 1) and 5 minutes (time 2) and 20 minutes after completion of the repeated sprint ability (time 3); (ii) 54 players were also tested for salivary testosterone in an unchallenged condition. We found that right-left 2D:4D was significantly and negatively related to testosterone concentrations at times 1, 2 and 3 following the repeated sprint agility test (P <0.05) and there was no association between the 2D:4D and basal testosterone levels in the unchallenged group. We suggest that low right-left 2D:4D is a predictive marker of free testosterone responsiveness when trained men are physically challenged, and that this association is programmed by the action of prenatal testosterone. © 2013 Copyright Taylor and Francis Group, LLC.