The optimal location for insertion of the transferred tendon in opposition transfer is controversial. The purpose of this study was to examine 4 commonly used insertion sites into the thumb and determine which maximizes thumb opposition. The flexor digitorum superficialis of the ring finger was used as a donor tendon and was attached in random order to the abductor pollicis brevis (APB) tendon, the APB and extensor pollicis longus, the flexor pollicis brevis (FPB) and dorsal radial extensor hood, and the ulnar extensor hood at the base of the proximal phalanx. As normal opposition was simulated, the minimum distance between the thumb and little finger and the pinch force were measured. The FPB and radial dorsal extensor hood site resulted in the statistically highest pinch force. The FPB and radial dorsal extensor hood and the APB sites had statistically smaller minimum distances between the thumb and little finger than the ulnar extensor hood site. A subjective evaluation of the 3-dimensional thumb path of motion revealed that the FPB and radial dorsal extensor hood site and the APB insertion site allowed the closest approximation of normal thumb opposition. This biomechanical study supports the use of the FPB and radial dorsal extensor hood insertion site or APB insertion site for opposition transfers.