The aim of this study was to evaluate the mechanical properties and biocompatibility of biomaterials, including bladder submucosa (BAMG), small intestinal submucosa (SIS), acellular corpus spongiosum matrix (ACSM), and polyglycolic acid (PGA), to identify the optimal scaffold for urethral tissue engineering. Tensile mechanical testing was conducted to evaluate mechanical properties of each scaffold. Rabbit corporal smooth muscle cells were cultured with the extracts of biomaterials and mitochondrial metabolic activity assay was used to determine the cytotoxicity of scaffold. The pore sizes of each scaffold were measured. Additionally, smooth muscle cells were seeded on biomaterials. Cell infiltration was evaluated. Mechanical evaluation showed that Young modulus, stress at break in ACSM were prior to those in other biomaterials (p < 0.05). MTT assay confirmed that all scaffolds supported normal cellular mitochondrial metabolic without inducing cytotoxic events. SEM demonstrated that PGA has the largest pore size (>200 microm). The ACSM has different pore sizes in urethral (<5 microm) and cavernosal surfaces (>10 microm). Widespread distribution of cells could be observed in PGA 14 days after seeding. Multilayer cellular coverage developed in BAMG and urethral surface of ACSM without any sign of cellular invasion. Moderated cellular penetration could be found in SIS and cavernosal surface of ACSM. Although each scaffold demonstrated suitable mechanical properties, which is similar to normal urethra, ACSM showed better response in some parameters than those in other biomaterials. It suggested that this scaffold may be an alternative for urethral reconstruction in the future. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.