Background Pseudoislet studies have concentrated on single beta-cell lines or a combination of insulin and glucagon-secreting cells, overlooking the potential role of somatostatin in insulin release. This study sought to evaluate a heterotypic pseudoislet model containing insulin- (MIN6), glucagon- (alpha TC1.9) and somatostatin (TGP52)-secreting cells of mouse origin and to compare these pseudoislets with traditional monolayer preparations. Methods Cellular viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays), proliferation (5-bromo-2-deoxyuridine ELISA), hormone content and functional insulin release in response to a variety of stimuli were measured. Differential expression of E-cadherin, connexin 36 and connexin 43 was assessed by reverse transcriptase-polymerase chain reaction and Western blot to determine a possible role for adherens in insulin release from these pseudoislets. Results All pseudoislet cells displayed reduced proliferation coupled with an increase in cell death which may contribute to their static size in culture. While MIN6 and TGP52 cells expressed E-cadherin and showed sustained or improved hormone content when configured as pseudoislets, alpha TC1.9 lacked E-cadherin and contained less glucagon following pseudoislet formation. MIN6 and alpha TC1.9 cells expressed connexin 36, but not connexin 43 and TGP52 cells expressed connexin 43 only. In the presence of Alanine, Arginine and glucagon-like peptide-1, heterotypic pseudoislet cultures secreted levels of insulin that were comparable to that of MIN6 pseudoislets. In addition, pseudoislets comprising all three cell lines released more insulin into the surrounding culture medium than MIN6 pseudoislets when studied over a 1-week period. Conclusions The current model may prove useful in studying the role of islet cell interactions in the release of insulin from pancreatic islets. Copyright (C) 2010 John Wiley & Sons, Ltd.