Reports that knee cartilage health is sensitive to kinematic changes, combined with reports of extension loss following ACL reconstruction, underscores the importance of restoring ambulatory knee extension in the context of preventing premature osteoarthritis. The purpose of this study was to test the relationship between individual variations in peak knee extension at heel-strike of walking and the anterior-posterior location of thickest cartilage in the medial and lateral femoral condyles of healthy contralateral and ACL reconstructed knees. In vivo gait analysis and knee MR images were collected from 29 subjects approximately 2 years after unilateral ACL reconstruction. Knee extension was measured at heel-strike of walking and 3-D femoral cartilage thickness models were reconstructed from MR images. The ACL reconstructed knees had significantly reduced knee extension (-1.5±4.2°) relative to the contralateral knees (-4.6±3.4°) at heel-strike of walking but did not have side-to-side differences in the anterior-posterior location or magnitude of thickest medial and lateral femoral cartilage. The anterior-posterior location of the thickest medial femoral cartilage was correlated with knee extension at heel-strike in both the healthy contralateral (R(2)=0.356, p<0.001) and reconstructed (R(2)=0.234, p=0.008) knees. These results suggest that ACL reconstruction can impair terminal extension at periods of ambulatory loading known to be related to cartilage morphology in healthy joints. The fact that the femoral cartilage thickness distribution had not changed at 2 years post-op, even in the subset of subjects with extension loss, suggests that loads may be shifted to thinner cartilage regions, which could have important implications on long-term joint health.