In vitro and in vivo analyses of stress on pacemaker leads and their components during the heart cycle have become especially important because of incidences of failure of some of these mechanical components. For stress analyses, the three-dimensional (3D) position, shape, and motion of the pacemaker leads must be known accurately at each time point during the cardiac cycle. We have developed a method for determination of the in vivo 3D positions of pacemaker leads during the entire heart cycle. Sequences of biplane images of patients with pacemakers were obtained at 30 frames/s for each projection. The sequences usually included at least two heart cycles. After patient imaging, biplane images of a calibration object were obtained from which the biplane imaging geometry was determined. The centerlines of the leads and unique, identifiable points on the attached electrodes were indicated manually for all acquired images. Temporal interpolation of the lead and electrode data was performed so that the temporal nonsynchronicity of the image acquisition was overcome. Epipolar lines, generated from the calculated geometry, were employed to identify corresponding points along the leads in the pairs of biplane images for each time point. The 3D positions of the lead and electrodes were calculated from the known geometry and from the identified corresponding points in the images. Using multiple image sets obtained with the calibration object at various orientations, the precision of the calculated rotation matrix and of the translation vector defining the imaging geometry was found to be approximately 0.7 degree and 1%, respectively. The 3D positions were reproducible to within 2 mm, with the error lying primarily along the axis between the focal spot and the imaging plane. Using data obtained by temporally downsampling to 15 frames/s, the interpolated data were found to lie within approximately 2 mm of the true position for most of the heart cycle. These results indicate that, with this technique, one can reliably determine pacemaker lead positions throughout the heart cycle, and thereby it will provide the basis for stress analysis on pacemaker leads.