The objectives of this study were (1) to develop subject-specific experimental and finite element (FE) techniques to study the three-dimensional stress-strain behavior of ligaments, with application to the human medial collateral ligament (MCL), and (2) to determine the importance of subject-specific material properties and initial (in situ) strain distribution for prediction of the strain distribution in the MCL under valgus loading. Eight male knees were subjected to varus-valgus loading at flexion angles of O", 30", and 60". Three-dimensional joint kinematics and MCL strains were recorded during kinematic testing. Following testing, the MCL of each knee was removed to allow measurement of the in situ strain distribution and to perform material testing. A FE model of the femur-MCL-tibia complex was constructed for each knee to simulate valgus loading at each flexion angle, using subject-specific bone and ligament geometry, material properties, and joint kinematics. A transversely isotropic hyperelastic material model was used to represent the MCL. The MCL in situ strain distribution at full extension was used to apply in situ strain to each MCL FE model. FE predicted MCL strains during valgus loading were compared to experimental measurements using regression analysis. The subject-specific FE predictions of strain correlated reasonably well with experimentally measured MCL strains (R' = 0.83, 0.72, and 0.66 at O", 30°, and 60", respectively). Despite large inter-subject variation in MCL material properties, MCL strain distributions predicted by individual FE models that used average MCL material properties were strongly correlated with subject-specific FE strain predictions (R' = 0.99 at all flexion angles). However, predictions by FE models that used average in situ strain distributions yielded relatively poor correlations with subject-specific FE predictions (R' = 0.44,0.35, and 0.33 at flexion angles of O", 30". and 60", respectively). The strain distribution within the MCL was nonuniform and changed with flexion angle. The highest MCL strains occurred at full extension in the posterior region of the MCL proximal to the joint line during valgus loading, suggesting this region may be most vulnerable to injury under these loading conditions. This work demonstrates that subject-specific FE models can predict the complex, nonuniform strain fields that occur in ligaments due to external loading of the joint.