AIMS/HYPOTHESIS: We assessed the heterogeneity of insulin secretion from human isolated beta cells and its regulation by cell-to-cell contacts. METHODS: Insulin secretion from single and paired cells was assessed by a reverse haemolytic plaque assay. The percentage of plaque-forming cells, the mean plaque area and the total plaque development were evaluated after 1 h of stimulation with different secretagogues. RESULTS: Not all beta cells were surrounded by a haemolytic plaque under all conditions tested. A small fraction of the beta cell population (20%) secreted more than 90% and 70% of total insulin at 2.2 and 22.2 mmol/l glucose, respectively. Plaque-forming cells, mean plaque area and total plaque development were increased at 12.2 and 22.2 compared with 2.2 mmol/l glucose. Insulin secretion of single beta cells was similar at 12.2 and 22.2 mmol/l glucose. Insulin secretion of beta cell pairs was increased compared with that of single beta cells and was higher at 22.2 than at 12.2 mmol/l glucose. Insulin secretion of beta cells in contact with alpha cells was also increased compared with single beta cells, but was similar at 22.2 compared with 12.2 mmol/l glucose. Delta and other non-beta cells did not increase insulin secretion of contacting beta cells compared with that of single beta cells. Differences in insulin secretion between 22.2 and 12.2 mmol/l glucose were observed in murine but not in human islets. CONCLUSIONS/INTERPRETATION: Human beta cells are highly heterogeneous in terms of insulin secretion so that a small fraction of beta cells contributes to the majority of insulin secreted. Homologous and heterologous intercellular contacts have a significant impact on insulin secretion and this could be related to the particular architecture of human islets.