The extensional behavior of tendon is closely linked to the directional organization of the collagen fibers in the tendon. We sought to demonstrate that quantitative information about this organization is accessible through the application of polarized light microscopy. Ten tendons (2 canine Achilles and 8 rat tail tendons) were sectioned in a longitudinal plane and stained with either hematoxylin and eosin or toluidine blue. One Achilles tendon was fixed without tension on the fibers, thus leaving the fibers in a wavy state, and the other was fixed while strained with longitudinal tension straightening the fibers. One hundred measurements of collagen fiber orientation were made on a 0.7 mm2 area from each tendon. Recorded orientation angles were plotted on circular histograms and analyzed using circular statistics. The strained fibers revealed a sharply peaked distribution, in contrast the unstrained fibers had a bimodal orientation distribution. The tail tendons were fixed at strains from 0-3%. Analysis of the orientation distributions revealed increasing alignment of the collagen as strain increased. These data, while matching the qualitative descriptions of the fibers, also provide quantitative assessment of organization enabling comparison among tissues of varied physical circumstances and function. This method of quantitative orientation measurement has wide application for the many birefringent tissues such as bone, smooth and striated muscle and elastin.