The soft-tissue interface between skin-mounted markers and the underlying bones poses a major limitation to accurate, non-invasive measurement of joint kinematics. The aim of this study was twofold: first, to quantify lower limb soft-tissue artifact in young healthy subjects during functional activity; and second, to determine the effect of soft-tissue artifact on the calculation of knee joint kinematics. Subject-specific bone models generated from magnetic resonance imaging (MRI) were used in conjunction with X-ray images obtained from single-plane fluoroscopy to determine three-dimensional knee joint kinematics for four separate tasks: open-chain knee flexion, hip axial rotation, level walking, and a step-up. Knee joint kinematics was derived using the anatomical frames from the MRI-based, 3D bone models together with the data from video motion capture and X-ray fluoroscopy. Soft-tissue artifact was defined as the degree of movement of each marker in the anteroposterior, proximodistal and mediolateral directions of the corresponding anatomical frame. A number of different skin-marker clusters (total of 180) were used to calculate knee joint rotations, and the results were compared against those obtained from fluoroscopy. Although a consistent pattern of soft-tissue artifact was found for each task across all subjects, the magnitudes of soft-tissue artifact were subject-, task- and location-dependent. Soft-tissue artifact for the thigh markers was substantially greater than that for the shank markers. Markers positioned in the vicinity of the knee joint showed considerable movement, with root mean square errors as high as 29.3mm. The maximum root mean square errors for calculating knee joint rotations occurred for the open-chain knee flexion task and were 24.3 degrees , 17.8 degrees and 14.5 degrees for flexion, internal-external rotation and abduction-adduction, respectively. The present results on soft-tissue artifact, based on fluoroscopic measurements in healthy adult subjects, may be helpful in developing location- and direction-specific weighting factors for use in global optimization algorithms aimed at minimizing the effects of soft-tissue artifact on calculations of knee joint rotations.