The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissue-engineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (+/-0.1) microg/mg compared to 0.1 (+/-0.1) microg/mg in decellularised bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility. Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery and tissue-engineering applications.