A tissue engineered Pancreatic substitute (TEPS) consisting of insulin-producing cells appropriately designed and encapsulated to support cellular function and prevent interaction with the host may provide physiological blood glucose regulation for the treatment of insulin dependent diabetes (IDD). The performance of agarose-based constructs which contained either a single cell suspension of GLUTag-INS cells, a suspension of pre-aggregated GLUTag-INS spheroids, or GLUTag-INS cells on small intestinal submucosa (SIS), was evaluated in vitro for total cell number, weekly glucose consumption and insulin secretion rates (GCR and ISR), and induced insulin secretion function. The three types of TEPS studied displayed similar number of cells, GCR, and ISR throughout 4 weeks of culture. However, the TEPS, which incorporated SIS as a substrate for the GLUTag-INS cells, was the only type of TEPS tested which was able to retain the induced insulin secretion function of non-encapsulated GLUTag-INS cells. Though improvements in the expression level of GLUTag-INS cells and/or the number of viable cells contained within the TEPS are needed for successful treatment of a murine model of IDD, this study has revealed a potential method for promoting proper cellular function of recombinant L-cells upon incorporation into an implantable three-dimensional TEPS. Biotechnol. Bioeng. 2009;103: 828-834. (c) 2009 Wiley Periodicals, Inc.