The new computerized imaging, circular polarized light microscopy technique was developed to measure the orientation of collagen fibers in images of serial sections of connective tissue. The system consists of a modified Olympus BX50 polarized microscope, a Sony AVC-D7 video camera, and a Silicon Graphics Indy computer. Both methods required the initial segmentation of fibers and used binary images. Segments of fiber midlines were traced with vertical and horizontal scanlines, or alternately the whole midlines were identified recursively from the Euclidean Distance Map of the image suing the novel definition of the Medial Axis Transform. The last technique produced connected midlines of the fibers and handled sinuous fibers well. The fiber midlines produced by this technique were traversed by a midline traversal algorithm , and the orientation distribution was obtained by least squares line fitting. The accuracy of the developed techniques was evaluated against synthetic images, composed of straight lines and sinuous curves. Kupier's statistic was used to evaluate the consistency of the fiber orientation calculations. Statistical analysis of the results showed, that the proposed Medial Axis Transform with Hilditch's connectivity preserving skeletonization produced the most accurate results. The developed method was used to measure collagen fiber orientation in microscopy images of canine meniscus, porcine aortic valve leaflet, bovine pericardium and bio- textiles.