Type 2 diabetes is characterized by islet amyloid deposits, which are primarily composed of the amyloidogenic human form of islet amyloid polypeptide (IAPP, amylin). The mechanism of islet amyloido-genesis is not known, but other products (e.g., apolipoprotein E and perlecan) contained within islet amyloid may be necessary. Because rodent IAPP does not form islet amyloid, the currently available beta-cell lines are not useful for studying processes involved in amyloid formation. To develop a suitable in vitro cell system for the study of islet amyloid formation, we generated two new beta-cell lines that express the amyloidogenic human IAPP. We did this by crossbreeding human IAPP transgenic mice with RIP-Tag mice that develop islet tumors and then culturing one of these islet tumors from two separate offspring of this cross. The resultant 2350-2C0 and 2511 cell lines produce human as well as mouse IAPP-like immunoreactivity (IAPP-LI) and immunoreactive insulin (IRI). Incubation of both these cell lines with 16.7 mmol/l glucose resulted in a two- to fourfold increase in human IAPP-LI, mouse IAPP-LI, and IRI secretion compared with 1.67 mmol/l glucose and the combination of 16.7 mmol/l glucose and 10 mmol/l arginine, 0.1 mmol/l 3-isobutyl-1-methylxanthine (IBMX), and 5 micromol/l carbachol induced a >50-fold increase in the release of these peptides. The omission of calcium from the above secretagogue cocktail reduced secretion of all three peptides to only two- to sixfold higher than the 16.7 mmol/l glucose condition. Perifusion with 16.7 mmol/l glucose plus 0.1 mmol/l IBMX caused a biphasic secretion of human IAPP-LI and mouse IAPP-LI, as well as IRI, in both cell lines, with the peak of the first phase being five- to sixfold higher than the prestimulated 1.67 mmol/l glucose condition. Immunoelectron microscopic inspection of both 2350-2C0 and 2511 cells after 7 days of culture did not reveal the presence of amyloid fibrils, suggesting the need for other critical components. We conclude that we have established two novel beta-cell lines that produce and secrete human IAPP in a regulated manner. These cell lines will be a useful tool to investigate the secretion of human IAPP as well as the necessity of other components for islet amyloid formation.