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This study asked whether the contribution of collagen V to the establishment of mechanical properties is tissue dependent. We mechanically tested four different tissues from wild type and targeted collagen V\u2010null mice: the flexor digitorum longus (FDL) tendon, Achilles tendon (ACH), the anterior cruciate ligament (ACL), and the supraspinatus tendon (SST). Area was significantly reduced in the <jats:italic>Col5a1<jats:sup>\u0394Ten\/\u0394Ten<\/jats:sup><\/jats:italic> group in the FDL, ACH, and SST. Maximum load and stiffness were reduced in the <jats:italic>Col5a1<jats:sup>\u0394Ten\/\u0394Ten<\/jats:sup><\/jats:italic> group for all tissues. However, insertion site and midsubstance modulus were reduced only for the ACL and SST. This study provides evidence that the regulatory role of collagen V in extracellular matrix assembly is tissue dependent and that joint instability in classic EDS may be caused in part by insufficient mechanical properties of the tendons and ligaments surrounding each joint. \u00a9 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. 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