Objective: Glucagon cell adenomatosis (GCA) was recently recognized by us as a multifocal neoplastic disease of the endocrine pancreas unrelated to MEN1. Multiple micro-and a few macrotumors are found on the background of a hyperplasia of glucagon cells. The disease may cause unspecific abdominal symptoms and only rarely a glucagonoma syndrome. Recently a mutation in the glucagon receptor (GCGR) gene was described in one GCA patient. Method: The extracted DNA of five patients with GCA was sequenced and the GCGR gene analyzed for mutations. Results: Sequencing of the GCGR gene revealed germline mutations in three out of five patients. One patient shows two different heterozygous point mutations in the hyperplastic alpha cells as well as in the non-tumorous tissue leading to two premature stop codons. One patient harbors a homozygous stop mutation. The third patient shows two homozygous missense mutations of the GCGR gene that most likely also led to a dysfunction of the GCGR. In the two other patients no germ line mutations of the GCGR gene were detected. These variants were not identified in healthy subjects. Conclusion: The finding of germ line and somatic "loss of function" mutations of the GCGR gene in three of five patients with GCA suggests that a change in the signalling function of the GCGR may cause glucagon cell adenomatosis via glucagon cell hyperplasia.