The action of the forearm is to transfer force and torque across the elbow and wrist, resulting in axial rotation at the distal and proximal radioulnar joints, called pronation and supination. Pronation and supination are important functions as they allow many kinds of activities of daily living. Three-dimensional (3D) motion analysis of pronation and supination is essential for a better understating of the biomechanics of the forearm to describe the physiological range of motion and optimize clinical options for disorders of the forearm. Although numerous in vitro studies have contributed to gain insights into the forearm pronation and supination, in vivo investigations are still limited. In this study, three-dimensional (3D) motion of the distal radioulnar joint (DRUJ) during forearm pronation and supination was analyzed in vivo using biplanar radiography and 3D models of the ulna and radius. Twelve healthy subjects (6 men and 6 women) were recruited for the study. Each subject's right forearm (dominant hand) underwent a computed tomography (CT) scan to create 3D surface models of the ulna and radius. Each subject was imaged using a calibrated biplanar radiographic system at 90° pronation, 45° pronation, neutral, 45° supination, and 90° supination while the elbow was in extension or flexion. The 3D positions of the radius and ulna were obtained using a 2D-to-3D image registration method. The relative translations of the radius in the radioulnar, dorsopalmar, and proximodistal directions and the relative rotations of the radius about the radioulnar, dorsopalmar, and proximodistal axes were evaluated with respect to the ulna during pronation and supination. In general, the radius translated towards the ulnar side throughout forearm rotation, and moved towards the palmar side as pronation increased and moved dorsally as supination increased, and was positioned proximally during pronation and distally during supination. The rotation of the radius was dominant about the proximodistal axis. These kinematic variables were affected by elbow position and sex of the subject. The current findings regarding the 3D kinematics of the DRUJ during forearm pronation and supination may contribute to further understanding of the physiological biomechanics of the upper limb.