Interactions between the endocrine cells in islets of Langerhans influence their secretory function, and disruption of islet structure results in impaired insulin secretory responses to both nutrient and non-nutrient stimuli. We have previously demonstrated that insulin-secreting MIN6 cells show enhanced secretory responses when grown as islet-like structures (pseudoislets) suggesting that homotypic cell–cell interactions between β-cells are important for normal function. We have now extended this experimental model to study the role of heterotypic interactions between insulin-expressing and glucagon-expressing cells by measuring the organization and secretory function of pseudoislets formed from MIN6 and αTC1 cells. The direct α-cell to β-cell contact in the heterogenous MIN6/αTC1 pseudoislets was sufficient to enable the formation of anatomically correct islet-like structures, with a central core of MIN6 cells surrounded by a periphery of αTC1 cells. However, the presence of αTC1 cells had no detectable effect on insulin secretory responses to nutrient or non-nutrient stimuli. In contrast, exogenous glucagon enhanced insulin secretion, in accordance with a paracrine role for α-cell-derived glucagon in the regulation of insulin secretion rather than direct, contact-mediated effects of α-cells on neighbouring β-cells.