The effect of long-chain free fatty acids (FFAs) on glucagon secretion from islet -cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in single -cells by fura-2 fluorescence imaging. OA at 30 µM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6-10 µM concentration-dependently increased [Ca²⁺]_i in -cells, irrespective of glucose concentrations (1.4, 2.8 and 8.3 mM). OA at 10 µM increased [Ca²⁺]_i in 90% of -cells. OA-induced [Ca²⁺]_i increases were strongly inhibited by the endoplasmic reticulum (ER) Ca²⁺-pump inhibitors, cyclopiazonic acid and thapsigargin, and by 2-aminoethoxydiphenyl borate (2-APB), the blocker of both IP3 receptors and store-operated Ca²⁺ channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca²⁺]_i increases was reduced substantially by Ca²⁺-free conditions and mildly by an L-type Ca²⁺ channel blocker nitrendipine and an ATP-sensitive K⁺ channel activator diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-APB. These results indicate that OA glucose-independently stimulate glucagon release by increasing [Ca²⁺]_i in rat pancreatic -cells and that the [Ca²⁺]_i increase is triggered by Ca²⁺ release from ER and amplified by Ca²⁺ influx possibly via store-operated channels and via voltage-dependent L-type Ca²⁺ channels. The glucose-independent action of OA to stimulate glucagon release from -cells may operate under hypoglycemic conditions when plasma FFAs levels are elevated, possibly playing a role in maintaining glucose metabolism.