We examined the transcriptional expression and functional effects of receptors for the extracellular pyrimidines uridine triphosphate (UTP) and uridine diphosphate (UDP), on insulin and glucagon secretion in isolated mouse pancreatic islets and purified β-cells. Using real-time PCR, the UDP receptor P2Y₆ was found to be highly expressed in both whole islets and β-cells purified by repeated counter-flow elutriation, whereas no mRNA expression for UTP receptors P2Y₄ and P2Y₂ could be detected. Functional in vitro experiments revealed that the P2Y₆ agonist UDPβS dose-dependently enhanced insulin and glucagon release during short-term incubation (1h), while P2Y₆ activation during a longer period (24h), selectively increased insulin release, especially at high glucose levels. The corresponding EC₅₀ value for UDPβS ranged from 3.2×10⁻⁸M to 1.6×10⁻⁸M for both glucose concentrations. The P2Y₆ antagonist MRS2578 inhibited the effects of UDPβS, supporting a P2Y₆ specific effect. In addition to negative RT-PCR results, the lack of response to UTPγS a selective P2Y_2/4 agonist further rule out the involvement of P2Y_2/4 receptors in the islet hormone release. Our results suggest a modulatory role for UDP via a functional active P2Y₆ receptor in the regulation of islet hormone release.