SIS (small intestinal submucosa) is a 3D extracellular matrix (ECM) material of porcine origin. It has a complex composition predominantly composed of collagen type I. SIS is rapidly absorbed, supports early and abundant new vessel growth, and serves as a template for the reconstructive remodelling of several body tissues. Currently SIS products are sterilised using ethylene oxide, gamma irradiation and e-beam irradiation. It is not known how they affect the materials properties such as structure, mechanical strength and biocompatibility. This study investigated the influence of each sterilisation method on the biocompatibility and biodegradation of SIS using L929 mouse fibroblasts. SIS samples were sterilised by each of the above methods under standard conditions. The samples were subjected to hydrolytic degradation conditions for specific periods of time. All sterilisation methods resulted in an increase in the rate of sample degradation. The study indicated that over time e-beam irradiation caused the greatest % weight loss. Applying sample extracts to L929 mouse fibroblasts assessed the biocompatibility of the degradation products. The % cellular protein and % metabolic activity were then assessed using the BCA assay and MTT assay, respectively. All SIS samples caused an increase in both cellular protein production and metabolic activity. Initially samples sterilised by ETO had the greatest effect but this decreased after 28 days. Unsterile samples were found to have a slower more prolonged influence. It is thought that the components released may include extractable growth factors and further studies are required to confirm this.