The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca²⁺ ([Ca²⁺]_c) in β-cells play a key role in the control of insulin secretion, and because oscillations of these two factors have been observed in single isolated islets and β-cells, pulsatile insulin secretion could theoretically result from [Ca²⁺]_c or metabolism oscillations. We could not detect metabolic oscillations independent from [Ca²⁺]_c changes in β-cells, and imposed metabolic oscillations were poorly effective in inducing oscillations of secretion when [Ca²⁺]_c was kept stable, which suggests that metabolic oscillations are not the direct regulator of the oscillations of secretion. By contrast, tight temporal and quantitative correlations between the changes in [Ca²⁺]_c and insulin release strongly suggest that [Ca²⁺]_c oscillations are the direct drivers of insulin secretion oscillations. Metabolism may play a dual role, inducing [Ca²⁺]_c oscillations (via changes in ATP-sensitive K⁺ channel activity and membrane potential) and amplifying the secretory response by increasing the efficiency of Ca²⁺ on exocytosis. The mechanisms underlying the oscillations of insulin secretion by the isolated pancreas and those observed in vivo remain elusive. It is not known how the functioning of distinct islets is synchronized, and the possible role of intrapancreatic ganglia in this synchronization requires confirmation. That pulsatile insulin secretion is beneficial in vivo, by preventing insulin resistance, is suggested by the greater hypoglycemic effect of exogenous insulin when it is infused in a pulsatile rather than continuous manner. The observation that type 2 diabetic patients have impaired pulsatile insulin secretion has prompted the suggestion that such dysregulation contributes to the disease and justifies the efforts toward understanding of the mechanism underlying the pulsatility of insulin secretion both in vitro and in vivo.