Cellular engineering studies in our group are directed at creating insulin-secreting cell lines that simulate the performance of the normal islet beta-cell. The strategy described in this article involves the stepwise stable introduction of genes relevant to beta-cell performance into the RIN 1046-38 insulinoma cell line, a process that we term "iterative engineering." RIN cells stably engineered to contain multiple copies of the human insulin gene exhibit a large increase in insulin content, such that they approach the content of human islets assayed in parallel. Analysis by high-performance liquid chromatography demonstrates that these engineered cell lines process human proinsulin to mature insulin with high efficiency. Cell lines that are further engineered to express the GLUT2 and glucokinase genes demonstrate stable expression of the three transgenes for the full lifetime of the lines produced to date (6 months to 1 year in continuous culture). Transplantation of the engineered cell lines into nude rats reveals that stably integrated genes are expressed at constant levels in the in vivo environment over the full duration of experiments performed (48 days). Several endogenous genes expressed in normal beta-cells, including rat insulin, amylin, sulfonylurea receptor, and glucokinase, are stably expressed in the insulinoma lines during these in vivo studies. Endogenous GLUT2 expression, in contrast, is rapidly extinguished during in vivo passage. The loss of GLUT2 is overcome in engineered cell ines in which transporter expression is provided by a stably transfected transgene. These results suggest that a potential advantage of the iterative engineering approach may be to preserve stability of function and phenotype, particularly in the in vivo setting.