Abstract Insulin secretion from MIN6 cells configured as cell aggregates by culture on a gelatin substrate (pseudoislets) is enhanced compared to that of MIN6 cells grown as monolayers on tissue culture plastic, indicating the importance of β-cell-to-β-cell proximity for insulin release. In this study we have shown that glucose induced a biphasic release of insulin from pseudoislets, whereas the amplitude and duration of the responses of equivalent monolayer cells were much reduced. Purinergic aqonists have been implicated in intercellular communication between β-cells, so we investigated whether adenine nucleotides co-released with insulin are responsible for the enhanced secretory responses of pseudoislets. We have demonstrated that MIN6 cells express purinergic A 1 and P2Y receptors, and that adenine nucleotides increased [Ca 2+] i with an efficacy of agonists being ATP>ADP>AMP. However, neither suramin nor the more selective A 1 antagonist 1,3-dipropyl-8-cyclopentylxanthine reduced glucose-induced insulin secretion from pseudoislets, and stimulation of monolayer cells with a range of adenine nucleotides did not enhance glucose-induced secretion. These results suggest that enhanced secretion from MIN6 pseudoislets is not due to increased paracrine/autocrine action of adenine nucleotides.