The mechanisms by which fatty acids increase insulin release are not known. In this study, mouse islets were used as a model and palmitate as a reference compound to study in vitro how saturated fatty acids influence pancreatic β-cells. Palmitate (625 μM) was bound to albumin. It did not affect basal insulin release (3 mM glucose) but increased the release induced by 10–15 mM glucose. This effect was dependent on the concentration of free rather than total palmitate. It was reversible and abolished by epinephrine, diazoxide, nimodipine, or omission of extracellular Ca. Bromopalmitate and methyl palmoxirate, two inhibitors of fatty acid oxidation, were ineffective alone, and only bromopalmitate partially inhibited the effects of palmitate on insulin release. The increase in insulin release produced by palmitate could not be ascribed to a blockade of ATP-sensitive K⁺-channels because the fatty acid only barely decreased ⁸⁶Rb efflux and did not depolarize β-cells in 3 mM glucose. The small effect on ⁸⁶Rb efflux might be attributed to a slight rise in the ATP/ADP ratio. No such rise occurred when palmitate was tested in 15 mM glucose, and the fatty acid consistently accelerated ⁸⁶Rb efflux under these conditions. Measurements of β-cell membrane potential (intracellular microelectrodes) and of free cytoplasmic calcium (Ca_i ²⁺) in β-cells (Fura 2 technique) showed that palmitate increases Ca²⁺ influx; it also caused a very small mobilization of intracellular Ca. The persistence of this stimulation of Ca²⁺ influx in the presence of diazoxide and high K⁺ suggests that palmitate might act on Ca²⁺ channels. The rise in Ca₁²⁺ produced by palmitate was accompanied by an increase in insulin release only if the concentration of glucose was sufficiently high. The β-cell response to palmitate thus differs from the responses to glucose and other metabolized nutrients in several respects. Saturated fatty acids appear to potentiate insulin release through an increase in Ca₁²⁺ and another, yet unidentified, fuel-dependent mechanism.