SUMMARYGiven the importance of thoraco-lumbar spine movements in the locomotion of mammals, it is surprising that in vivo threedimensional (3-D) data on the intervertebral movement of the mammalian thoraco-lumbar vertebral column during symmetrical gaits is limited to horses and dogs. To test whether kinematic patterns similar to those published for these cursorial species are also present during a contrasting mode of quadrupedalism, we quantified thoraco-lumbar intervertebral movements, the resulting pelvic displacements and relative femoral movements during the trot-like steady-state suspensory quadrupedal locomotion of the two-toed sloth (Xenarthra, Choloepus didactylus). Scientific rotoscoping, a new, non-invasive approach that combines synchronous biplanar high speed X-ray videos and the reconstruction of skeletal elements from computed tomography bone scans, was used to quantify 3-D kinematics. An analysis of vertebral anatomy and epaxial muscle topography suggests that the thoraco-lumbar spine of sloths is well suited to producing lateral bending and long-axis rotation, but limits powerful sagittal extension. Sloths exhibit complex 3-D movements in the thoraco-lumbar spine that are comparable to those observed in other arboreal quadrupedal mammals. Monophasic lateral bending and long-axis rotation, biphasic sagittal bending and maximal amplitude of sagittal bending at the lumbo-sacral joint were also found in other quadruped mammals and may represent general aspects of mammalian symmetric gaits. Maximal amplitude of lateral bending and long-axis rotation vary in regard to the vertebral level. It is suggested that a cranio-caudal pattern of angular deflections of the spine results from the out-of-phase movement of diagonal forelimbs and hindlimbs in other walking gaits, because it is not evident in the trot-like locomotion analyzed here. The analysis also illustrates the difficulties that arise when lumbar movement is deduced from intervertebral joint morphology alone.