The effects of increasing concentrations of leucine (0.2, 2.0, and 15.0 mmol/liter) on glucagon secretion from the perfused rat pancreas were examined at various glucose levels (0, 3.3, or 8.3 mmol/liter) and in the absence or presence of either arginine (5.0 mmol/liter) or glutamine (10.0 mmol/liter). At a low glucose concentration (3.3 mmol/liter), leucine caused a dose-related biphasic increase in glucagon output in the absence of arginine, but only a transient increase in the presence of the latter amino acid. These positive responses were markedly reduced and, on occasion, abolished at a high glucose concentration (8.3 mmol/liter). Moreover, at a low glucose concentration (3.3 mmol/liter) and in the presence of arginine, the highest concentration of leucine (15.0 mmol/liter) provoked a sustained and reversible inhibition of glucagon release. Likewise, leucine (15.0 mmol/liter) reversibly inhibited glucagon secretion evoked by glutamine in the absence of glucose. Thus, leucine exerted a dual effect on the secretion of glucagon, the inhibitory effect of leucine prevailing at a high concentration of the branched chain amino acid and when glucagon secretion was already stimulated by arginine or glutamine. At a physiological concentration (0.2 mmol/liter), however, leucine was a positive stimulus for glucagon release, especially in the absence of another amino acid. Concomitantly, leucine was always a positive stimulus for both insulin and somatostatin secretion. The intimate mechanisms involved in the dual effect of leucine on glucagon secretion remain to be elucidated.