The ability to measure cyclic changes in myocardial sarcomere lengths and orientations during cardiac ejection and filling would improve our understanding of how the cellular processes of contraction relate to the pumping of the whole heart. Previously, only postmortem sarcomere measurements were possible after arresting the heart in one state and fixing it for histology. By combining such histological measurements with direct observations of the deformation experienced by the same myocardial region while the heart was beating, we have developed a method to reconstruct sarcomere lengths and orientations throughout the cardiac cycle and at several transmural layers. A set of small (1 mm) radiopaque beads was implanted in approximately 1 cm3 of the left ventricular free wall. Using biplane cineradiography, we tracked the motion of these markers through various cardiac cycles. To quantify local myocardial deformation (as revealed by the relative motion of the markers), we calculated the local deformation gradient tensors. As the heart deforms, these describe how any short vectorial line segment alters its length and orientation relative to a reference state. Specifically, by choosing the reference state to be the arrested and fixed heart and by measuring the sarcomere vector in that state, we could then use the deformation gradient tensors to reconstruct the sarcomere vector that would exist in the beating heart. As ventricular chamber volume varied over its normal range of operation, the range of reconstructed sarcomere lengths (approximately 1.7-2.4 microns) was comparable to other histological studies and to measurements of sarcomere length in excised papillary muscles or trabeculae. The pattern of sarcomere length changes was markedly different, however, during ejection vs. filling.