New growth of extraocular muscle has been demonstrated in degenerating peripheral nerve autografts implanted between two extraocular muscles. This suggests that extraocular muscle may be lengthened for therapeutic purposes if a suitable matrix can be found to support this new growth. Investigators of peripheral nerve regeneration have found that the basal lamina of freeze-killed skeletal muscle remains intact and supports axonal regeneration. This study was designed to investigate the feasibility of inducing regenerative growth of extraocular muscle in freeze-treated extraocular muscle autografts. In six beagles the inferior oblique muscle was removed from both orbits, frozen in liquid nitrogen, and allowed to thaw at room temperature. The freeze-thaw cycle was repeated. The freeze-treated grafts were then sewn in an end-to-end fashion between the cut end of the lateral rectus and the globe. At both 4 and 8 weeks postoperatively, three dogs were killed, and the grafts were removed from both orbits. These were prepared for light and electron microscopic examination. This revealed robust growth of mature-appearing, innervated muscle fibers in the proximal graft that could be differentiated by adenosine triphosphatase histochemistry. Rare, immature fibers were seen in the distal graft. These results demonstrate that freeze-treated extraocular muscle autografts support regenerative growth of extraocular muscle.