Sphingosine (10 microM) induced mobilization of intracellular Ca2+ stores in the pancreatic duct adenocarcinoma cell line CFPAC-1. The effect was specific for sphingosine, since the sphingosine analog C2-ceramide had no effect. Sphingosine did not cause Ca2+ entry from extracellular medium, as also shown by following Mn2+ quenching of Fura-2 fluorescence. Furthermore, sphingosine, similarly to the mitochondrial inhibitors rotenone and oligomycin, strongly inhibited the rate of Mn2+ entry triggered by both thapsigargin- and agonist-induced depletion of intracellular stores. The uptake of rhodamine 123, a lipophilic cation which estimates mitochondrial energy level, was reduced by sphingosine to an extent similar to that observed in the presence of mitochondrial inhibitors. It is suggested that impairment of mitochondrial function might be responsible for inhibition of capacitative Ca2+ entry caused by sphingosine.