The effect of cell-to-cell contact on Ca2+ influx and secretory responses in the beta-cell line MIN6 was studied using MIN6 pseudoislets, which are three-dimensional islet-like cell aggregates that develop when MIN6 cells are cultured for 6-8 days on gelatin. The formation of pseudoislets is dependent on the Ca2+-dependent adhesion molecule E-cadherin (E-CAD), since the process can be inhibited by incubation in the absence of Ca2+ or in the presence of an anti-E-CAD antibody. Glucose and alpha-ketoisocaproic acid (KIC) evoked a Ca2+ influx in only a small fraction of the MIN6 monolayer cells, whereas >80% of cell groups within the pseudoislets responded to both nutrients. In contrast, changes in the intracellular free Ca2+ concentration ([Ca2+]i) were observed in all or most monolayer cells or pseudoislet cell groups in response to physical or pharmacological depolarizing stimuli. No significant increase in insulin release was observed from MIN6 monolayer cells in response to nutrient or nonnutrient insulin secretagogues. Conversely, pseudoislets were found to respond significantly to both nutrients and nonnutrients. These results suggest that close cell-to-cell contact improves the functional responsiveness of MIN6 cells and that pseudoislets may therefore serve as a useful research model in the study of beta-cell function.