Although numerous studies have addressed the effects of ACL injury and reconstruction on knee joint motion, there is currently little data available describing in vivo ACL strain during activities of daily living. Data describing in vivo ACL strain during activities such as gait is critical to understanding the biomechanical function of the ligament, and ultimately, to improving the surgical treatment of patients with ACL rupture. Thus, our objective was to characterize the relative strain in the ACL during both the stance and swing phases of normal level walking. Eight normal subjects were recruited for this study. Through a combination of magnetic resonance imaging, biplanar fluoroscopy, and motion capture, we created in vivo models of each subject's normal walking movements to measure knee flexion, ACL length, and relative ACL strain during gait. Regression analysis demonstrated an inverse relationship between knee flexion and ACL length (R(2)=0.61, p<0.001). Furthermore, relative strain in the ACL peaked at 13±2% (mean±95%CI) during mid-stance when the knee was near full extension. Additionally, there was a second local maximum of 10±7% near the end of swing phase, just prior to heel strike. These data are a vital step in further comprehending the normal in vivo biomechanics experienced by the ACL. In the future, this information could prove critical to improving ACL reconstruction and provide useful validation to future computational models investigating ACL function.