Pancreatic β-cells from obese-hyperglycemic (ob/ob) mice are widely used for studying the mechanisms of insulin release, including its regulation by the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In this study, we compared changes of [Ca²⁺]_i in single β-cells isolated from ob/ob mice with those from lean mice using dual-wavelength microfluorometry and the indicator fura-2. There were no differences in the frequency, amplitude, and half-width of the slow oscillations induced by glucose. Most β-cells from the obese mice responded to 10 mM caffeine with transformation of the oscillations into sustained elevation of [Ca²⁺]_i, a process counteracted by ryanodine. The β-cells from the obese mice were characterized by ample generation of [Ca²⁺]_i transients, which increased in number in the presence of glucagon. The transients became less frequent when leptin was added at a concentration as low as 1 nM. It is suggested that the excessive firing of [Ca²⁺]_i transients in the ob/ob mice is owing to the absence of leptin and is mediated by activation of the phospholipase C signaling pathway.