Free fatty acids (FFA) have been proposed to participate in the regulation of insulin release from pancreatic beta-cells (beta-cells). As a rise in cytosolic free Ca2+ ([Ca(2+)]i) is a key event for the stimulation of insulin secretion, the effects of saturated FFA on [Ca2+]i were investigated. Palmitate was used as a reference compound and [Ca2+]i was measured in single fura-2 loaded HIT-T15 and in primary mouse beta-cells. Stimulation of single beta-cells with palmitate (100 microM) caused either repetitive Ca2+ transients or a plateau-like rise in [Ca2+]i. In HIT-T15 and in mouse beta-cells, the number of palmitate-responsive cells, and the amplitude of the palmitate-induced Ca2+-signals were dependent on the extracellular glucose concentration. In Ca2+-free medium palmitate (100 microM) caused only 1 or 2 Ca2+ transients indicating mobilization of Ca2+ from internal stores. Withdrawal of external Ca2+, the addition of voltage-sensitive Ca2+ channel (VSCC) blockers, as well as the K(ATP)-channel opener diazoxide (100 microM) reversibly blocked the palmitate-induced cytosolic Ca2+ responses. This demonstrates that Ca2+ influx through VSCC of the L-type coupled to membrane depolarization through closure of K(ATP)-channels are crucial for a sustained Ca2+-signal in response to palmitate. Methyl palmoxirate (100 microM) and 2-bromopalmitate (100 microM), which both inhibit transport of acyl-CoA into the mitochondria, reversibly blocked the palmitate-induced Ca2+-signals in HIT-T15 as well as in primary mouse beta-cells. By contrast, cerulenin (100 microM), an inhibitor of protein acylation, had no effect on the palmitate-induced changes in [Ca2+]i, which suggests that mitochondrial palmitate metabolism is required for eliciting the Ca2+-signals. Simultaneous measurement of [Ca2+]i and the mitochondrial membrane potential (DeltaPsi) revealed palmitate-induced depolarization of DeltaPsi 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 Ca2+-signals in pancreatic beta-cells.