While free fatty acids (FFA) are well known as insulin secretagogues, their effects on pancreatic α cells have been mostly neglected. In the present study we therefore systematically analyzed the glucagon metabolism of rat pancreatic islets under the influence of FFA. Primary islets were incubated in the presence or absence of 200 μmol/L albumin-complexed palmitate or oleate at 2.8 mmol/L versus 16.7 mmol/L glucose and glucagon secretion was monitored over 8 hours. In addition to these time-course experiments, dose dependency of palmitate-induced effects was tested by a 2-hour incubation with 50 to 300 μmol/L albumin-complexed palmitate at 2.8 mmol/L and 5.6 mmol/L glucose. Apart from glucagon secretion, intracellular immunoreactive glucagon and cellular preproglucagon-mRNA (PPG-mRNA) content were determined from the remaining cell lysates. FFA, especially palmitate, induced a significant and dose-dependent increase of glucagon secretion (in average 2-fold above control) during the first 120 minutes of incubation at low to normal glucose (2.8 and 5.6 mmol/L). There was no significant glucagonotropic effect of FFA at concomitant 16.7 mmol/L glucose. Intracellular glucagon as well as cellular PPG-mRNA content were found to be dose-dependently diminished by palmitate when compared with untreated controls at 5.6 mmol/L glucose. The present analysis therefore points to a new role for FFA as a nutritient secretagogue and a modulator of α-cellular glucagon metabolism.