External ATP is believed to initiate and propagate Ca²⁺ signals co-ordinating the insulin release pulses within and among the different islets in the pancreas. The possibility that islet endothelial cells participate in this process was evaluated by comparing the effects on [Ca²⁺]_i of purinoceptor activation in these cells with those in β-cells. β-Cell-rich pancreatic islets were isolated from ob/ob mice and dispersed into single cells/aggregates. After culture with or without endothelial cell growth supplement (ECGS), the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was measured with ratiometric fura-2 technique. Presence of ECGS or prolongation of culture (>5 days) resulted in proliferation of endothelial cells and altered their phenotype from rounded to elongated. Endothelial cells, preliminarily identified by attachment of Dynabeads coated with the Bandeiraea simplicifolia 1 lectin (BS-1), responded in a similar way as those stained with CD31 antibodies after measurements of [Ca²⁺]_i. Spontaneous transients and oscillations of [Ca²⁺]_i were seen in β-cells, but not in endothelial cells exposed to 20 mM glucose. Addition of ATP (10 μM) resulted in pronounced and more extended rise of [Ca²⁺]_i in endothelial cells than in β-cells. The endothelial cells differed from the β-cells by also responding with a rise of [Ca²⁺]_i to 10 μM UTP, but not to equimolar ADP and acetylcholine. The results support the idea of mutual interactions between islet endothelium and β-cells based on ATP-induced Ca²⁺ signals. It is suggested that the endothelial cells have a tonic inhibitory action on β-cell P2 purinoceptors resulting in impaired synchronization of the insulin release pulses.