The vascular architecture of pancreatic islets is complex, and its blood flow regulation is autonomous from that of the exocrine pancreas. To separately study islet vascular reactivity we established a novel technique enabling us to isolate and perfuse islet arterioles. For this purpose, islets with their arterioles were microdissected from normoglycemic and alloxan-diabetic C57BL/6 mice and then perfused in a chamber at a pressure-adjusted flow rate of approximately 40 nl/min, aiming at a pressure of 40 mmHg in the arterioles. The arteriolar diameters could be continuously registered and measured. The diameter of the studied of islets was 400-600 mm and the arteriole diameters were 40 mm. D-glucose, the main insulin secretagogue, had a selective dilating effect on vascular smooth muscle in islet arterioles, but not in glomerular afferent arterioles. Furthermore, this response was amplified in islet arterioles prepared from diabetic animals. Angiotensin II (Ang II) and L–NAME contracted islet arterioles, whereas adenosine dilated them. The constrictive effects of Ang II on islet arterioles were concentration-dependent and very pronounced at higher concentrations. However, the sensitivity was much less than in glomerular afferent arterioles. Pretreatment with L-NAME and spermine NONO-ate prevented Ang II-mediated vasoconstriction in islet arterioles. The study presents, for the first time, a technique which enables us to isolate and perfuse single pancreatic islets, which will provide unique opportunities for further studies on the regulation of islet blood perfusion.