In order to investigate the three-dimensional structure of the collagen fibrils in articular cartilage, full-thickness canine humeral cartilage was microtomed into perpendicular sections that included both the articular surface and the subchondral bone and approximately 100 successive parallel sections that were each 6 microm thick and from a different cartilage depth. Each section was imaged using polarized light microscopy with a 5x objective (2.0 microm pixel size), generating two quantitative images (angle and retardation). Selected sections were also imaged using a 40x objective (0.25 microm pixel size). At an increased depth from the articular surface, the angle and retardation results in the perpendicular sections showed the well-known 90 degrees change in fibril orientation between the surface and the deep cartilage. In contrast, the retardation results of the parallel sections decreased from the articular surface and remained approximately 0 through most of the radial zones, while the angle results of the parallel sections only changed about 30 degrees. The territorial matrix morphology surrounding 61 chondrocyte clusters was quantified by its length, aspect ratio, and orientation. The cellular clusters in the surface cartilage were ellipsoidal in both parallel and perpendicular sections. In the radial zone, the cellular clusters were oriented in vertical columns in the perpendicular sections and as circular groupings in the parallel sections. This orthogonal imaging technique could provide a better understanding of the three-dimensional territorial and interterritorial fibrils in articular cartilage, the disturbance of which could signify the onset of degenerative cartilage diseases such as osteoarthritis.