Penile conditions, such as Peyronie's disease or tumor resection may require surgical reconstruction of the tunica albuginea. Various materials have been proposed, as a biomaterial for tunica albuginea repair, however, little functional data are available. We examined the applicability and functional outcome of a collagen-based matrix derived from the bladder (acellular bladder matrix (ABM)), as a biomaterial for tunica repair. Biocompatibility testing was performed on the matrix, which included mitochondrial metabolic activity, cell viability and apoptosis. Approximately 50% of the dorsal penile tunica albuginea was replaced with the collagen-based matrix patch after surgical removal in 24 New Zealand White rabbits. Cavernosometry and cavernosography were performed. The animals were killed 1, 2 and 3 months after surgery for analyses. The matrix showed excellent biocompatibility. All animals implanted with the matrix survived without any noticeable untoward effects. There was no evidence of inflammation or infection at the time of retrieval. Cavernosometry of the implanted animals demonstrated normal intracavernosal pressures with visual erections. Cavernosography of the repaired corpora showed a normal anatomical configuration. Biomechanical analysis of the retrieved matrices demonstrated similar tensile strengths as native tunica. Histologically, there was only a minimal inflammatory response, which gradually decreased over time. These results show that ABM is biocompatible, durable and effective when used as a tunica substitute. The matrix may be useful as an off-the-shelf biomaterial for patients requiring tunica albuginea repair.