Injuries to the anterior cruciate ligament (ACL) result in immediate and long-term morbidity and expense. Young women are more likely to sustain ACL injuries than men who participate in similar athletic and military activities. Although significant attention has focused on the role that fe male sex hormones may play in this disparity, it is still unclear if the female ACL also responds to androgens. The purpose of this study was to determine if the female ACL was an androgen responsive tissue. In order to identify and localize androgen receptors in the female ACL, we used western blotting and immunofluorescent labeling, respectively, of ACL tissue harvested during surgery from young females (n = 3). We then measured ACL stiffness and assessed total (T) and free (FAI) testosterone concentrations, as well as relative estradiol to testosterone ratios (E2:T and E2:FAI) at three consecutive menstrual stages (n = 20). There were significant rank order correlations between T (0.48, p=0.031), FAI (0.44, p=0.053), E2:T (-0.71, p<0.001), E2:FAI (-0.63, p=0.003) ratios, and ACL stiffness near ovulation. With the influences of the other variables controlled, there were significant negative partial rank order correlations between ACL stiffness and the E2:T (-0.72, p<0.001) and E2:FAI (-0.59, p=0.012) ratios. The partial order residuals for T and FAI were not significant. These findings suggest that the female ACL is an androgen responsive tissue, but that T and FAI are not independent predictors of ACL stiffness near ovulation. Instead, the relations hip between T, FAI, and ACL stiffness was likely influenced by another hormone or sex hormone binding globulin.