Free fatty acids (FFA) have been proposed to participate in the regulation of insulin release from pancreatic beta-cells (β-cells). As a rise in cytosolic free Ca 2+ ([Ca 2+] i ) is a key event for the stimulation of insulin secretion, the effects of saturated FFA on [Ca 2+] i were investigated. Palmitate was used as a reference compound and [Ca 2+] i was measured in single fura-2 loaded HIT-T15 and in primary mouse β-cells. Stimulation of single β-cells with palmitate (100 μM) caused either repetitive Ca 2+ transients or a plateau-like rise in [Ca 2+] i . In HIT-T15 and in mouse β-cells, the number of palmitate-responsive cells, and the amplitude of the palmitate-induced Ca 2+-signals were dependent on the extracellular glucose concentration. In Ca 2+-free medium palmitate (100 μM) caused only 1 or 2 Ca 2+ transients indicating mobilization of Ca 2+ from internal stores. Withdrawal of external Ca 2+, the addition of voltage-sensitive Ca 2+ channel (VSCC) blockers, as well as the K ATP-channel opener diazoxide (100 μM) reversibly blocked the palmitate-induced cytosolic Ca 2+ responses. This demonstrates that Ca 2+ influx through VSCC of the L-type coupled to membrane depolarization through closure of K ATP-channels are crucial for a sustained Ca 2+-signal in response to palmitate. Methyl palmoxirate (100 μM) and 2-bromopalmitate (100 μM), which both inhibit transport of acyl-CoA into the mitochondria, reversibly blocked the palmitate-induced Ca 2+-signals in HIT-T15 as well as in primary mouse β-cells. By contrast, cerulenin (100 μM), an inhibitor of protein acylation, had no effect on the palmitate-induced changes in [Ca 2+] i , which suggests that mitochondrial palmitate metabolism is required for eliciting the Ca 2+-signals. Simultaneous measurement of [Ca 2+] i and the mitochondrial membrane potential (Δ Ψ) revealed palmitate-induced depolarization of Δ Ψ which demonstrates that palmitate does not enhance mitochondrial ATP production. Therefore mitochondrial signals other than ATP appear to be generated from palmitate metabolism that underly the palmitate-induced Ca 2+-signals in pancreatic β-cells.